Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

PubMed

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

A Molecular Approach Designed to Limit the Replication of Mature DENV2 in Host Cells.

PubMed

Raheel, Ummar; Jamal, Muhsin; Zaidi, Najam Us Sahar Sadaf

2015-09-01

Dengue virus (DENV) is an arthropod-borne virus, which belongs to the Flaviviridae family, and completes its life cycle in two hosts: humans and mosquitoes. For DENV maturation, the surface pre-membrane (prM) protein is cleaved to form a mature membrane protein (M) by furin, which is a cellular enzyme subsequently releasing the mature virus from the host dendritic cell. The objective of the current study was to inhibit mature DENV isotype 2 (DENV2) by RNA-interference in a Vero-81 cell line. Mature DENV2 was propagated in and isolated from U937 cells expressing dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin. Maturation of DENV2 was confirmed by Western blot analysis, where virus stock lacking prM was considered mature. Inhibition studies were carried out by transfection of Vero-81 cells with six synthetic siRNAs along with a control siRNA. Reduction in cellular DENV2 was observed also by focus-reduction assay, immunofluorescence assay (IFA), and real-time quantitative polymerase chain reaction (RT-qPCR). Cells transfected with DENV2SsiRNA2, which was targeting the structural region M of mature DENV2, was able to reduce DENV2 titer by up to 85% in focus reduction assays. A significant reduction in mature DENV2 RNA load was observed by RT-qPCR, confirming the previous findings. IFA also revealed reduced levels of cellular DENV2. These results demonstrated that mature DENV2 can be effectively inhibited by synthetic siRNA targeting the structural region of the genome. Mature DENV2 can be successfully inhibited by siRNAs, and specifically high knock-down efficiency is observed by siRNAs against M region of mature DENV2. This study shows that M represents a potential target for RNAi based inhibitory approaches.

Back to the Thymus: Peripheral T Cells Come Home

PubMed Central

Hale, J. Scott; Fink, Pamela J.

2009-01-01

The thymus has long been known as the generative organ for the T cell arm of the immune system. To perform this role, the thymus was thought to require protection from antigenic and cellular insult from the “outside world”, with the notable exception of the continual influx of progenitor cells required to initiate the complicated process of T cell differentiation. Overwhelming evidence that mature T cells can recirculate and persist in the thymus has required us to revamp this earlier view of the thymus as detached from outside influence. In this review, we consider the evidence for T cell recirculation into the thymus, discuss the likely means and location of mature T cell entry, and speculate on the potential consequences of such close apposition between differentiating thymocytes and mature recirculating lymphocytes. PMID:19030016

Back to the thymus: peripheral T cells come home.

PubMed

Hale, J Scott; Fink, Pamela J

2009-01-01

The thymus has long been known as the generative organ for the T-cell arm of the immune system. To perform this role, the thymus was thought to require protection from antigenic and cellular insult from the 'outside world', with the notable exception of the continual influx of progenitor cells required to initiate the complicated process of T-cell differentiation. Overwhelming evidence that mature T cells can recirculate and persist in the thymus has required us to revamp this earlier view of the thymus as detached from outside influence. In this review, we consider the evidence for T-cell recirculation into the thymus, discuss the likely means and location of mature T-cell entry, and speculate on the potential consequences of such close apposition between differentiating thymocytes and mature recirculating lymphocytes.

Calcium Signaling enhancement during oocyte maturation

NASA Astrophysics Data System (ADS)

Jung, Peter; Ullah, Ghanim; Machaca, Khaled

2006-03-01

A Ca2+ signal with a special spatial and temporal characteristic universally removes cell-cycle arrest after fertilization of a mature egg cell. The Ca2+ signal is characterized by a fast rise of intracellular Ca2+ and a slow decay on the time scale of minutes. We use computational modeling of Ca2+ release on the microscale (Ca2+ puffs) and cell-scale in conjunction with experimental knowledge of the changes in the Ca2+ signaling apparatus during oocyte maturation and changing signaling patterns to explore the relationship between organization and sensitivity of IP3 receptors and SERCA pumps and the resulting signaling patterns. We hypothesize that potentiation of the IP3 receptors during oocyte maturation is the main cause for the differentiation in the signaling patterns.

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen

PubMed Central

Tian, Chaorui; Yuan, Xueli; Jindra, Peter T.; Bagley, Jessamyn; Sayegh, Mohamed H.; Iacomini, John

2010-01-01

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation. PMID:20452826

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

PubMed Central

Mills, Richard J.; Titmarsh, Drew M.; Koenig, Xaver; Parker, Benjamin L.; Ryall, James G.; Quaife-Ryan, Gregory A.; Voges, Holly K.; Hodson, Mark P.; Ferguson, Charles; Drowley, Lauren; Plowright, Alleyn T.; Needham, Elise J.; Wang, Qing-Dong; Gregorevic, Paul; Xin, Mei; Thomas, Walter G.; Parton, Robert G.; Nielsen, Lars K.; Elliott, David A.; Porrello, Enzo R.

2017-01-01

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies. PMID:28916735

A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia

NASA Astrophysics Data System (ADS)

Managò, Stefano; Valente, Carmen; Mirabelli, Peppino; Circolo, Diego; Basile, Filomena; Corda, Daniela; de Luca, Anna Chiara

2016-04-01

Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

Control of the rate of cell enlargement: Excision, wall relaxation, and growth-induced water potentials.

PubMed

Boyer, J S; Cavalieri, A J; Schulze, E D

1985-04-01

A new guillotine thermocouple psychrometer was used to make continuous measurements of water potential before and after the excision of elongating and mature regions of darkgrown soybean (Glycine max L. Merr.) stems. Transpiration could not occur, but growth took place during the measurement if the tissue was intact. Tests showed that the instrument measured the average water potential of the sampled tissue and responded rapidly to changes in water potential. By measuring tissue osmotic potential (Ψ s ), turgor pressure (Ψ p ) could be calculated. In the intact plant, Ψ s and Ψ p were essentially constant for the entire 22 h measurement, but Ψ s was lower and Ψ p higher in the elongating region than in the mature region. This caused the water potential in the elongating region to be lower than in the mature region. The mature tissue equilibrated with the water potential of the xylem. Therefore, the difference in water potential between mature and elongating tissue represented a difference between the xylem and the elongating region, reflecting a water potential gradient from the xylem to the epidermis that was involved in supplying water for elongation. When mature tissue was excised with the guillotine, Ψ s and Ψ p did not change. However, when elongating tissue was excised, water was absorbed from the xylem, whose water potential decreased. This collapsed the gradient and prevented further water uptake. Tissue Ψ p then decreased rapidly (5 min) by about 0.1 MPa in the elongating tissue. The Ψ p decreased because the cell walls relaxed as extension, caused by Ψ p , continued briefly without water uptake. The Ψ p decreased until the minimum for wall extension (Y) was reached, whereupon elongation ceased. This was followed by a slow further decrease in Y but no additional elongation. In elongating tissue excised with mature tissue attached, there was almost no effect on water potential or Ψ p for several hours. Nevertheless, growth was reduced immediately and continued at a decreasing rate. In this case, the mature tissue supplied water to the elongating tissue and the cell walls did not relax. Based on these measurements, a theory is presented for simultaneously evaluating the effects of water supply and water demand associated with growth. Because wall relaxation measured with the psychrometer provided a new method for determining Y and wall extensibility, all the factors required by the theory could be evaluated for the first time in a single sample. The analysis showed that water uptake and wall extension co-limited elongation in soybean stems under our conditions. This co-limitation explains why elongation responded immediately to a decrease in the water potential of the xylem and why excision with attached mature tissue caused an immediate decrease in growth rate without an immediate change in Ψ p.

In vitro effects of ambroxol on Cryptococcus adherence, planktonic cells, and biofilms.

PubMed

Kong, Qingtao; Du, Xue; Huang, Suyang; Yang, Rui; Zhang, Chengzhen; Shen, Yongnian; Liu, Weida; Sang, Hong

2017-07-01

The antifungal effects of ambroxol (Amb; the metabolite VIII of bromhexine) against Cryptococcus planktonic cells and mature biofilms were investigated in this study. Amb showed antifungal activity against planktonic cells and mature biofilms. Disk diffusion test similarly showed antifungal profile for planktonic cells. Furthermore, Amb was found to be synergetic with fluconazole against planktonic cells and reduced the adherence of cells to polystyrene. Our results suggest that Amb can inhibit cryptococcal cells and biofilms, indicating its potential role in the prevention and treatment of cryptococcosis. © 2017 APMIS. Published by John Wiley & Sons Ltd.

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

NASA Astrophysics Data System (ADS)

Luchinat, Enrico; Barbieri, Letizia; Rubino, Jeffrey T.; Kozyreva, Tatiana; Cantini, Francesca; Banci, Lucia

2014-11-01

Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone.

Role of ataxia-telangiectasia mutated (ATM) in porcine oocyte in vitro maturation.

PubMed

Lin, Zi-Li; Kim, Nam-Hyung

2015-06-01

Ataxia-telangiectasia mutated (ATM) is critical for the DNA damage response, cell cycle checkpoints, and apoptosis. Significant effort has focused on elucidating the relationship between ATM and other nuclear signal transducers; however, little is known about the connection between ATM and oocyte meiotic maturation. We investigated the function of ATM in porcine oocytes. ATM was expressed at all stages of oocyte maturation and localized predominantly in the nucleus. Furthermore, the ATM-specific inhibitor KU-55933 blocked porcine oocyte maturation, reducing the percentages of oocytes that underwent germinal vesicle breakdown (GVBD) and first polar body extrusion. KU-55933 also decreased the expression of DNA damage-related genes (breast cancer 1, budding uninhibited by benzimidazoles 1, and P53) and reduced the mRNA and protein levels of AKT and other cell cycle-regulated genes that are predominantly expressed during G2/M phase, including bone morphogenetic protein 15, growth differentiation factor 9, cell division cycle protein 2, cyclinB1, and AKT. KU-55933 treatment decreased the developmental potential of blastocysts following parthenogenetic activation and increased the level of apoptosis. Together, these data suggested that ATM influenced the meiotic and cytoplasmic maturation of porcine oocytes, potentially by decreasing their sensitivity to DNA strand breaks, stimulating the AKT pathway, and/or altering the expression of other maternal genes. © 2015 International Federation for Cell Biology.

Pre-malignant lymphoid cells arise from hematopoietic stem/progenitor cells in chronic lymphocytic leukemia.

PubMed

Kikushige, Yoshikane; Miyamoto, Toshihiro

2015-11-01

Human malignancies progress through a multistep process that includes the development of critical somatic mutations over the clinical course. Recent novel findings have indicated that hematopoietic stem cells (HSCs), which have the potential to self-renew and differentiate into multilineage hematopoietic cells, are an important cellular target for the accumulation of critical somatic mutations in hematological malignancies and play a central role in myeloid malignancy development. In contrast to myeloid malignancies, mature lymphoid malignancies, such as chronic lymphocytic leukemia (CLL), are thought to originate directly from differentiated mature lymphocytes; however, recent compelling data have shown that primitive HSCs and hematopoietic progenitor cells contribute to the pathogenesis of mature lymphoid malignancies. Several representative mutations of hematological malignancies have been identified within the HSCs of CLL and lymphoma patients, indicating that the self-renewing long-lived fraction of HSCs can serve as a reservoir for the development of oncogenic events. Novel mice models have been established as human mature lymphoma models, in which specific oncogenic events target the HSCs and immature progenitor cells. These data collectively suggest that HSCs can be the cellular target involved in the accumulation of oncogenic events in the pathogenesis of mature lymphoid and myeloid malignancies.

Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

NASA Astrophysics Data System (ADS)

Zheng, Xiaofei; Zhou, Fengjuan; Gu, Yifei; Duan, Xiaobo; Mo, Anchun

2017-02-01

Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells.

Mature Hepatocytes Exhibit Unexpected Plasticity by Direct Dedifferentiation into Liver Progenitor Cells in Culture

PubMed Central

Chen, Yixin; Wong, Philip P.; Sjeklocha, Lucas; Steer, Clifford J.; Sahin, M. Behnan

2011-01-01

Although there have been numerous reports describing the isolation of liver progenitor cells from adult liver, their exact origin has not been clearly defined; and the role played by mature hepatocytes as direct contributors to the hepatic progenitor cell pool has remained largely unknown. Here we report strong evidence that mature hepatocytes in culture have the capacity to dedifferentiate into a population of adult liver progenitors without genetic or epigenetic manipulations. By using highly-purified mature hepatocytes, which were obtained from untreated, healthy rat liver and labeled with fluorescent dye PKH2, we found that hepatocytes in culture gave rise to a population of PKH2-positive liver progenitor cells. These cells, Liver Derived Progenitor Cells or LDPCS, which share phenotypic similarities with oval cells, were previously reported to be capable of forming mature hepatocytes both in culture and in animals. Studies done at various time points during the course of dedifferentiation cultures revealed that hepatocytes rapidly transformed into liver progenitors within one week through a transient oval cell-like stage. This finding was supported by lineage-tracing studies involving double-transgenic AlbuminCreXRosa26 mice expressing β-galactosidase exclusively in hepatocytes. Cultures set up with hepatocytes obtained from these mice resulted in generation of β-galactosidase-positive liver progenitor cells demonstrating that they were a direct dedifferentiation product of mature hepatocytes. Additionally, these progenitors differentiated into hepatocytes in vivo when transplanted into rats that had undergone retrorsine pretreatment and partial hepatectomy. Conclusion Our studies provide strong evidence for the unexpected plasticity of mature hepatocytes to dedifferentiate into progenitor cells in culture; and this may potentially have a significant impact on the treatment of liver diseases requiring liver or hepatocyte transplantation. PMID:21953633

Reversal of hippocampal neuronal maturation by serotonergic antidepressants

PubMed Central

Kobayashi, Katsunori; Ikeda, Yumiko; Sakai, Atsushi; Yamasaki, Nobuyuki; Haneda, Eisuke; Miyakawa, Tsuyoshi; Suzuki, Hidenori

2010-01-01

Serotonergic antidepressant drugs have been commonly used to treat mood and anxiety disorders, and increasing evidence suggests potential use of these drugs beyond current antidepressant therapeutics. Facilitation of adult neurogenesis in the hippocampal dentate gyrus has been suggested to be a candidate mechanism of action of antidepressant drugs, but this mechanism may be only one of the broad effects of antidepressants. Here we show a distinct unique action of the serotonergic antidepressant fluoxetine in transforming the phenotype of mature dentate granule cells. Chronic treatments of adult mice with fluoxetine strongly reduced expression of the mature granule cell marker calbindin. The fluoxetine treatment induced active somatic membrane properties resembling immature granule cells and markedly reduced synaptic facilitation that characterizes the mature dentate-to-CA3 signal transmission. These changes cannot be explained simply by an increase in newly generated immature neurons, but best characterized as “dematuration” of mature granule cells. This granule cell dematuration developed along with increases in the efficacy of serotonin in 5-HT4 receptor-dependent neuromodulation and was attenuated in mice lacking the 5-HT4 receptor. Our results suggest that serotonergic antidepressants can reverse the established state of neuronal maturation in the adult hippocampus, and up-regulation of 5-HT4 receptor-mediated signaling may play a critical role in this distinct action of antidepressants. Such reversal of neuronal maturation could affect proper functioning of the mature hippocampal circuit, but may also cause some beneficial effects by reinstating neuronal functions that are lost during development. PMID:20404165

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole.

PubMed

Lee, Bo Yon; Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo

2011-11-18

In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue. Copyright © 2011 Elsevier Inc. All rights reserved.

Numerically bridging lamellipodial and filopodial activity during cell spreading reveals a potentially novel trigger of focal adhesion maturation.

PubMed

Loosli, Y; Vianay, B; Luginbuehl, R; Snedeker, J G

2012-05-01

We present a novel approach to modeling cell spreading, and use it to reveal a potentially central mechanism regulating focal adhesion maturation in various cell phenotypes. Actin bundles that span neighboring focal complexes at the lamellipodium-lamellum interface were assumed to be loaded by intracellular forces in proportion to bundle length. We hypothesized that the length of an actin bundle (with the corresponding accumulated force at its adhesions) may thus regulate adhesion maturation to ensure cell mechanical stability and morphological integrity. We developed a model to test this hypothesis, implementing a "top-down" approach to simplify certain cellular processes while explicitly incorporating complexity of other key subcellular mechanisms. Filopodial and lamellipodial activities were treated as modular processes with functional spatiotemporal interactions coordinated by rules regarding focal adhesion turnover and actin bundle dynamics. This theoretical framework was able to robustly predict temporal evolution of cell area and cytoskeletal organization as reported from a wide range of cell spreading experiments using micropatterned substrates. We conclude that a geometric/temporal modeling framework can capture the key functional aspects of the rapid spreading phase and resultant cytoskeletal complexity. Hence the model is used to reveal mechanistic insight into basic cell behavior essential for spreading. It demonstrates that actin bundles spanning nascent focal adhesions such that they are aligned to the leading edge may accumulate centripetal endogenous forces along their length, and could thus trigger focal adhesion maturation in a force-length dependent fashion. We suggest that this mechanism could be a central "integrating" factor that effectively coordinates force-mediated adhesion maturation at the lamellipodium-lamellum interface.

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Bo Yon, E-mail: boyonlee@gmail.com; Shim, Sang Woo; Kim, Young Sun

Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice andmore » in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.« less

Developmental potential of elongating and elongated spermatids obtained after in-vitro maturation of isolated round spermatids.

PubMed

Cremades, N; Sousa, M; Bernabeu, R; Barros, A

2001-09-01

Round spermatid injections are associated with disappointing clinical outcomes, and although these cells have been shown to mature into late spermatids in vitro, the developmental potential of such gametes remains to be demonstrated. Round spermatids were isolated from 12 testicle samples of patients with obstructive azoospermia, hypoplasia, complete maturation arrest, and incomplete Sertoli cell-only syndrome. They were cultured for 7 days at 32 degrees C, 5% CO(2)in air, in microdrops of Vero cell-conditioned medium containing 10% synthetic serum substitute. From the 238 round spermatids cultured, 25.2% attained the elongating and 5.5% the elongated spermatid stage (3-4 days per step). Relatively higher maturation rates were found in cases with obstructive azoospermia, but differences were significant only for elongated spermatids (9.3%). No differences were found in maturation rates between cases with non-obstructive azoospermia (4.3% of elongated spermatids). Experimental microinjections with elongating and elongated spermatids revealed a low fertilization rate (40.9%) but a normal blastocyst formation rate (60%). Late spermatids resulting from in-vitro culture of round spermatids in conditioned medium, either in controls in cases with a spermiogenetic block, appeared able to successfully fertilize the human oocyte and elicit normal embryo development.

Interaction between human mature adipocytes and lymphocytes induces T-cell proliferation.

PubMed

Poloni, Antonella; Maurizi, Giulia; Ciarlantini, Marco; Medici, Martina; Mattiucci, Domenico; Mancini, Stefania; Maurizi, Angela; Falconi, Massimo; Olivieri, Attilio; Leoni, Pietro

2015-09-01

Adipose tissue is a critical organ that plays a major role in energy balance regulation and the immune response through intricate signals. We report on the inter-relation between mature adipocytes and lymphocytes in terms of adipocyte-derived T-cell chemo-attractants and adipocyte metabolic effects on lymphocytes. During the culture time, mature adipocytes changed their structural and functional properties into de-differentiated cells. Isolated mature adipocytes expressed significantly higher levels of CIITA, major histocompatibility complex II (human leukocyte antigen [HLA]-DR) and costimulatory signal molecule CD80 compared with adipocytes after the de-differentiation process. Moreover, human leukocyte antigen-G, which may prevent the immune responses of mesenchymal stromal cells, was expressed at lower level in mature adipocytes compared with de-differentiated adipocytes. In line with these molecular data, functional results showed different immunoregulatory properties between adipocytes before and after the de-differentiation process. Mature adipocytes stimulated the proliferation of total lymphocytes and immunoselected cell populations CD3+, CD4+ and CD8+ in a direct contact-dependent way that involved the major histocompatibility complex I and II pathways. Moreover, adipocytes secreted potential chemo-attractant factors, but data showed that adipocyte-derived culture medium was not sufficient to activate lymphocyte proliferation, suggesting that a direct contact between adipocytes and immune cells was needed. However, specific mature adipocyte cytokines enhanced lymphocyte proliferation in a mixed lymphocyte reaction. In conclusion, cross-talk occurs between adipocytes and lymphocytes within adipose tissue involving T-cell chemo-attraction by mature adipocytes. Our findings, together with current observations in the field, provide a rationale to identify adipocyte-lymphocyte cross-talk that instigates adipose inflammation. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Maturation of neuronal form and function in a mouse thalamo-cortical circuit.

PubMed

Warren, R A; Jones, E G

1997-01-01

Postnatal development of physiological properties underlying slow intrathalamic oscillations was studied by whole-cell recording from synaptically coupled neurons of the reticular nucleus (RTN) and ventral posterior nucleus (VPN) of mouse brain slices in vitro and compared with the morphological development of dye-injected cells. Between postnatal days 3 and 11 (P3-P11), progressive changes in RTN and VPN neurons included shortening of the membrane time constant, decreasing input resistance, and lowering of the resting membrane potential (RMP). Low-threshold Ca2+ spikes (LTS) were present from P3, but their capacity to sustain multispike bursts was limited before P11. Synaptic responses were evoked in RTN and VPN neurons by electrical stimulation of the internal capsule from P3. Younger RTN neurons responded with a single spike, but their capacity to fire bursts gradually improved as the RMP reached levels below the LTS activation potential. Concomitantly, as the reversal potential of the inhibitory postsynaptic potential in VPN neurons became more negative, its capacity to deinactivate the LTS increased, and rebound bursts that could maintain oscillations were produced; sustained oscillations became the typical response to internal capsule stimulation at P12. The functional maturation of the intrathalamic circuitry, particularly between P10 and P14, occurs in parallel with the morphological maturation (size, dendritic growth, and dendritic field structure) of individual RTN and VPN neurons, as studied by confocal microscopy. Maturation of RTN cells led that of VPN cells by 2-3 d. The appearance of intrathalamic oscillations is probably correlated with the appearance of slow-wave sleep in postnatal animals.

A method for deriving homogenous population of oligodendrocytes from mouse embryonic stem cells.

PubMed

Neman, J; de Vellis, J

2012-06-01

There is a pressing need for new therapeutics for the generation and transplantation of oligodendrocyte to the white matter to help replace and render injured cells that are lost in demyelinating disease. There are a few protocols describing a homogenous derivation of non-manipulated mouse embryonic stem cells to oligodendrocytes (ES-OL). Moreover, protocols that are successful in producing ES-OL do so with low efficiency. Therefore, we describe clear methodology for differentiation of mouse ES cells to oligodendrocyte to a high degree of homogenity and reproducibility in vitro. In addition, taking advantage of three defined media, we can generate a defined ES to oligodendrocyte lineage while selecting against neurons and astrocytes. More specifically, (1) Glial stem cell defining media (GSCDM), supplemented with appropriate combination of SHH and RA support pro-oligodendrocyte developing neural spheres from ES cells, (2) Oligodendrocyte differentiating media, induces lineage selection of oligodendrocytes progenitors from neural stem cells, and (3) Oligodendrocyte maturation media, supports oligodendrocytes progenitor maturation. Moreover, the ES cell derived oligodendrocytes display mature properites in the prescence of rat dorsal root gangila in vitro. Thus confirming thier potential for use to invesitgate developmental pathways and future potential use of cells in transplantation towards myelin repair. Copyright © 2012 Wiley Periodicals, Inc.

Potential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures.

PubMed

Sandström, J; Broyer, A; Zoia, D; Schilt, C; Greggio, C; Fournier, M; Do, K Q; Monnet-Tschudi, F

2017-05-01

Exposure to environmental toxicants during vulnerable windows of brain development is suspected to raise the prevalence for neurological dysfunctions at later stages in life. Differentiation processes and changes in morphology, as well as a lack of physiological barriers, might be reasons that render a developing brain more susceptible to neurotoxicants than an adult. However, also the intrinsic capacity of cells to combat toxicant induced cellular stress might differ between the immature- and mature brain. In order to study whether this intrinsic protection capacity differs between immature and maturated brain cells we chose to study the maturation-dependent adverse effects of the known neurotoxicant Paraquat Dichloride (PQ) in 3D rat brain cell cultures. This in vitro system consists of the major brain cell types - neurons, astrocytes, oligodendrocytes and microglia - and over the time in vitro cultured cells undergo differentiation and maturation into a tissue-like organization. PQ was applied repeatedly over ten days in the sub-micromolar range, and effects were evaluated on neurons and glial cells. We observed that despite a higher PQ-uptake in mature cultures, PQ-induced adverse effects on glutamatergic-, GABAergic- and dopaminergic neurons, as assessed by gene expression and enzymatic activity, were more pronounced in immature cultures. This was associated with a stronger astrogliosis in immature- as compared to mature cultures, as well as perturbations of the glutathione-mediated defense against oxidative stress. Furthermore we observed evidence of microglial activation only in mature cultures, whereas immature cultures appeared to down-regulate markers for neuroprotective M2-microglial phenotype upon PQ-exposure. Taken together our results indicate that immature brain cell cultures have less intrinsic capacity to cope with cellular stress elicited by PQ as compared to mature cells. This may render immature brain cells more susceptible to the adverse effects of PQ. Copyright © 2017 Elsevier B.V. All rights reserved.

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

DOE PAGES

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; ...

2015-10-17

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

A regulatory role for TGF-β signaling in the establishment and function of the thymic medulla.

PubMed

Hauri-Hohl, Mathias; Zuklys, Saulius; Holländer, Georg A; Ziegler, Steven F

2014-06-01

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-β in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-β signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.

Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation.

PubMed

Sati, Leyla; Ovari, Laszlo; Bennett, David; Simon, Stephen D; Demir, Ramazan; Huszar, Gabor

2008-04-01

Individual spermatozoa were assessed with pairs of probes for persistent histones and cytoplasmic retention, persistent histones and DNA fragmentation, and persistent histones and apoptotic markers. The individual spermatozoa were treated sequentially with combinations of probes for these cytoplasmic and nuclear biochemical markers. Sperm fields were recorded with computer-assisted imaging, and staining patterns with the two probes in the same spermatozoa were examined and scored as light, intermediate or dark (mature to arrested-maturity spermatozoa). The effects of arrested sperm maturation were similar with respect to the cytoplasmic and nuclear characteristics of spermatozoa in 84% of cells, indicating that cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, there were moderate (intermediate-dark or intermediate-light patterns, 14.5% of cells) or major (light-dark patterns, 1.6% of cells) discrepancies in the intensity of the double staining patterns. Thus, testing with single maturity markers may not be fully reliable. These findings are important with respect to: (i) arrested sperm maturation; (ii) potential efficacy of antioxidant and similar therapeutic strategies in subfertile men, as spermatozoa with infrastructure defects due to mismaturation or maturation arrest are unlikely to respond to interventions; and (iii) detection of adverse male environmental exposures.

Xenopus laevis oocyte maturation is affected by metal chlorides.

PubMed

Marin, Matthieu; Slaby, Sylvain; Marchand, Guillaume; Demuynck, Sylvain; Friscourt, Noémie; Gelaude, Armance; Lemière, Sébastien; Bodart, Jean-François

2015-08-01

Few studies have been conducted using Xenopus laevis germ cells as oocytes, though these cells offer many advantages allowing both electrophysiological studies and morphological examination. Our aim was to investigate the effects of metal (cadmium, lead, cobalt and zinc) exposures using cell biology approaches. First, cell survival was evaluated with both phenotypical and electrophysiological approaches. Secondly, the effect of metals on oocyte maturation was assessed with morphological observations and electrophysiological recordings. From survival experiments, our results showed that metal chlorides did not affect cell morphology but strongly depolarized X. laevis oocyte resting potential. In addition, cadmium chloride was able to inhibit progesterone-induced oocyte maturation. By contrast, zinc, but also to a lesser extent cadmium, cobalt and lead, were able to enhance spontaneous oocyte maturation in the absence of progesterone stimulation. Finally, electrophysiological recordings revealed that some metal chlorides (lead, cadmium) exposures could disturb calcium signaling in X. laevis oocyte by modifying calcium-activated chloride currents. Our results demonstrated the high sensitivity of X. laevis oocytes toward exogenous metals such as lead and cadmium. In addition, the cellular events recorded might have a predictive value of effects occurring later on the ability of oocytes to be fertilized. Together, these results suggest a potential use of this cellular lab model as a tool for ecotoxicological assessment of contaminated fresh waters. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human natural killer cell development in secondary lymphoid tissues

PubMed Central

Freud, Aharon G.; Yu, Jianhua; Caligiuri, Michael A.

2014-01-01

For nearly a decade it has been appreciated that critical steps in human natural killer (NK) cell development likely occur outside of the bone marrow and potentially necessitate distinct microenvironments within extramedullary tissues. The latter include the liver and gravid uterus as well as secondary lymphoid tissues such as tonsils and lymph nodes. For as yet unknown reasons these tissues are naturally enriched with NK cell developmental intermediates (NKDI) that span a maturation continuum starting from an oligopotent CD34+CD45RA+ hematopoietic precursor cell to a cytolytic mature NK cell. Indeed despite the detection of NKDI within the aforementioned tissues, relatively little is known about how, why, and when these tissues may be most suited to support NK cell maturation and how this process fits in with other components of the human immune system. With the discovery of other innate lymphoid subsets whose immunophenotypes overlap with those of NKDI, there is also need to revisit and potentially re-characterize the basic immunophenotypes of the stages of the human NK cell developmental pathway in vivo. In this review, we provide an overview of human NK cell development in secondary lymphoid tissues and discuss the many questions that remain to be answered in this exciting field. PMID:24661538

Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus.

PubMed

Gomez, M P; Segura, J

1996-08-01

Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants.

ALA-PDT mediated DC vaccine for skin squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Ji, Jie; Fan, Zhixia; Zhou, Feifan; Wang, Xiaojie; Shi, Lei; Zhang, Haiyan; Wang, Peiru; Yang, Degang; Zhang, Linglin; Wang, Xiuli; Chen, Wei R.

2015-03-01

Dendritic cell (DC) based vaccine has emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have only achieved limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secret INF-Υ and IL-12). ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumor in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing DC-based cancer vaccine, which could improve the clinical application of PDT- DC vaccines.

The effects of Candida albicans cell wall protein fraction on dendritic cell maturation.

PubMed

Roudbary, Maryam; Roudbar Mohammadi, Shahla; Bozorgmehr, Mahmood; Moazzeni, Seyed Mohammad

2009-06-01

Candida albicans is a member of the normal human microflora. C. albicans cell wall is composed of several protein and carbohydrate components which have been shown to play a crucial role in C. albicans interaction with the host immune system. Major components of C. albican cell wall are carbohydrates such as mannans, beta glucans and chitins, and proteins that partially modulate the host immune responses. Dendritic cells (DC), as the most important antigen-presenting cells of the immune system, play a critical role in inducing immune responses against different pathogens. We investigated the effect of the cell wall protein fraction (CPF) of C. albicans on DC maturation. The CPF of C. albicans cells was extracted by a lysis buffer containing sodium dodecyl sulphate, 2-mercaptoethanol and phosphate-buffered saline. The extract was dialyzed and its protein pattern was evaluated by electrophoresis. Dendritic cells were purified from Balb/c mice spleens through a three-step method including mononuclear cell separation, as well as 2-h and overnight cultures. The purified CPF was added at different concentrations to DC. The purity and maturation status of DC were determined by flow cytometry using monoclonal antibodies against CD11c, MHC-II, CD40 and CD86. Treatment of DC with 10 microg/ml of CPF increased the expression of maturation markers including MHC-II, CD86 and CD40 on DC compared to the control group. In this study we used C. albicans CPF with the molecular weight of 40-45 kDa for pulsing and maturation of dendritic cells. Since according to our results CPF significantly increased the expression of maturation markers on DC, we suggest that CPF may act as an efficient immunomodulator, or may be used as a potential adjuvant to boost the host immune system against infections.

Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo.

PubMed

Phadnis, Smruti M; Joglekar, Mugdha V; Dalvi, Maithili P; Muthyala, Sudhakar; Nair, Prabha D; Ghaskadbi, Surendra M; Bhonde, Ramesh R; Hardikar, Anandwardhan A

2011-03-01

The scarcity of human islets for transplantation remains a major limitation of cell replacement therapy for diabetes. Bone marrow-derived progenitor cells are of interest because they can be isolated, expanded and offered for such therapy under autologous/allogeneic settings. We characterized and compared human bone marrow-derived mesenchymal cells (hBMC) obtained from (second trimester), young (1-24 years) and adult (34-81 years) donors. We propose a novel protocol that involves assessment of paracrine factors from regenerating pancreas in differentiation and maturation of hBMC into endocrine pancreatic lineage in vivo. We observed that donor age was inversely related to growth potential of hBMC. Following in vitro expansion and exposure to specific growth factors involved in pancreatic development, hBMC migrated and formed islet-like cell aggregates (ICA). ICA show increased abundance of pancreatic transcription factors (Ngn3, Brn4, Nkx6.1, Pax6 and Isl1). Although efficient differentiation was not achieved in vitro, we observed significant maturation and secretion of human c-peptide (insulin) upon transplantation into pancreactomized and Streptozotocin (STZ)-induced diabetic mice. Transplanted ICA responded to glucose and maintained normoglycemia in diabetic mice. Our data demonstrate that hBMC have tremendous in vitro expansion potential and can be differentiated into multiple lineages, including the endocrine pancreatic lineage. Paracrine factors secreted from regenerating pancreas help in efficient differentiation and maturation of hBMC, possibly via recruiting chromatin modulators, to generate glucose-responsive insulin-secreting cells.

Surface Modified Biodegradable Electrospun Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

PubMed

Sorkio, Anni; Porter, Patrick J; Juuti-Uusitalo, Kati; Meenan, Brian J; Skottman, Heli; Burke, George A

2015-09-01

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications.

Developmental potential of murine germinal vesicle stage cumulus-oocyte complexes following exposure to dimethylsulphoxide or cryopreservation: loss of membrane integrity of cumulus cells after thawing.

PubMed

Ruppert-Lingham, C J; Paynter, S J; Godfrey, J; Fuller, B J; Shaw, R W

2003-02-01

Cumulus cells of the cumulus-oocyte complex (COC) are important in oocyte maturation. Thus, in preserving immature oocytes it is prudent to also preserve their associated cumulus cells. The survival and function of oocytes and their associated cumulus cells was assessed following cryopreservation or exposure to cryoprotectant without freezing. Immature COCs were collected from mice primed with pregnant mare's serum. COCs were either slow-cooled or exposed to 1.5 mol/l dimethylsulphoxide without freezing. Treated and fresh COCs were stained for membrane integrity or, after in-vitro maturation and IVF, were assessed for developmental capability. Development of cumulus-denuded fresh oocytes, as well as denuded and frozen-thawed oocytes co-cultured with fresh cumulus cells, was assessed. Slow-cooled oocytes had significantly reduced coverage by intact cumulus cells compared with fresh COCs. Cumulus cell association and developmental capability were not substantially affected by exposure to cryoprotectant without freezing. Denuded fresh oocytes and cryopreserved COCs had decreased developmental potential that was not overcome by co-culture with fresh cumulus cells. Loss of association between oocyte and cumulus cells was induced by cryopreservation, but not by treatment with cryoprotectant alone. The data indicate that direct physical contact between cumulus cells and the oocyte, throughout maturation, improves subsequent embryo development.

Treatment with specific soluble factors promotes the functional maturation of transcription factor-mediated, pancreatic transdifferentiated cells.

PubMed

Motoyama, Hiroaki; Kobayashi, Akira; Yokoyama, Takahide; Shimizu, Akira; Sakai, Hiroshi; Notake, Tsuyoshi; Fukushima, Kentaro; Miyagawa, Shin-Ichi

2018-01-01

Pancreatic lineage-specific transcription factors (TFs) display instructive roles in converting adult cells to endocrine pancreatic cells through a process known as transdifferentiation. However, little is known about potential factors capable of accelerating transdifferentiation following transduction to achieve the functional maturation of transdifferentiated cells. In this study, we demonstrated, using adult liver-derived progenitor cells, that soluble factors utilized in pancreatic differentiation protocols of pluripotent stem cells promote functional maturation of TFs-mediated transdifferentiated cells. Treatment with an N2 supplement in combination with three soluble factors (glucagon-like peptide-1 [GLP-1] receptor agonist, notch inhibitor, and transforming growth factor-β [TGF-β] inhibitor) enhanced liver-to-pancreas transdifferentiation based on the following findings: i) the incidence of c-peptide-positive cells increased by approximately 1.2-fold after the aforementioned treatment; ii) the c-peptide expression level in the treated cells increased by approximately 12-fold as compared with the level in the untreated cells; iii) the treated cells secreted insulin in a glucose-dependent manner, whereas the untreated cells did not; and iv) transplantation of treated-transdifferentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the amelioration of hyperglycemia. These results suggest that treatment with specific soluble factors promotes the functional maturation of transdifferentiated cells. Our findings could facilitate the development of new modalities for cell-replacement therapy for patients with diabetes.

Expression of CTLA-4 (CD152) on human medullary CD4+ thymocytes.

PubMed

Castan, J; Klauenberg, U; Kalmár, P; Fleischer, B; Bröker, B M

1998-06-01

CTLA-4 (CD152) is a T cell surface receptor with sequence homology to the co-stimulatory molecule CD28. The molecule, which is essential for the inhibitory regulation of the immune response, becomes transiently expressed on mature T cells after stimulation in vitro. In situ, CTLA-4+ T cells are enriched in the light zones of the germinal centers in human peripheral lymphoid organs. In this study we have studied expression of CTLA-4 in human thymus in situ. CTLA-4 was expressed on about one third of CD4+/CD8-/CD1- medullary thymocytes. CTLA-4 was acquired by a subset of immature (CD1+) thymocytes and lost from the mature (CD1-) subpopulation within 48 h of cell culture, suggesting that the expression on medullary thymocytes is transient. The demonstration of CTLA-4 on a substantial subpopulation of mature CD4+ thymocytes adds a new dimension to the understanding of this important molecule. When contemplating application of anti-CTLA-4 for therapy its potential influence on T cell maturation has to be taken into account.

Paracrine Maturation and Migration of SH-SY5Y Cells by Dental Pulp Stem Cells.

PubMed

Gervois, P; Wolfs, E; Dillen, Y; Hilkens, P; Ratajczak, J; Driesen, R B; Vangansewinkel, T; Bronckaers, A; Brône, B; Struys, T; Lambrichts, I

2017-06-01

Neurological disorders are characterized by neurodegeneration and/or loss of neuronal function, which cannot be adequately repaired by the host. Therefore, there is need for novel treatment options such as cell-based therapies that aim to salvage or reconstitute the lost tissue or that stimulate host repair. The present study aimed to evaluate the paracrine effects of human dental pulp stem cells (hDPSCs) on the migration and neural maturation of human SH-SY5Y neuroblastoma cells. The hDPSC secretome had a significant chemoattractive effect on SH-SY5Y cells as shown by a transwell assay. To evaluate neural maturation, SH-SY5Y cells were first induced toward neuronal cells, after which they were exposed to the hDPSC secretome. In addition, SH-SY5Y cells subjected to the hDPSC secretome showed increased neuritogenesis compared with nonexposed cells. Maturated cells were shown to increase immune reactivity for neuronal markers compared with controls. Ultrastructurally, retinoic acid (RA) signaling and subsequent exposure to the hDPSC secretome induced a gradual rise in metabolic activity and neuronal features such as multivesicular bodies and cytoskeletal elements associated with cellular communication. In addition, electrophysiological recordings of differentiating cells demonstrated a transition toward a neuronal electrophysiological profile based on the maximum tetrodotoxin (TTX)-sensitive, Na + current. Moreover, conditioned medium (CM)-hDPSC-maturated SH-SY5Y cells developed distinct features including, Cd 2+ -sensitive currents, which suggests that CM-hDPSC-maturated SH-SY5Y acquired voltage-gated Ca 2+ channels. The results reported in this study demonstrate the potential of hDPSCs to support differentiation and recruitment of cells with neuronal precursor characteristics in a paracrine manner. Moreover, this in vitro experimental design showed that the widely used SH-SY5Y cell line can improve and simplify the preclinical in vitro research on the molecular mechanisms of stem cell-mediated neuronal regeneration.

Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

PubMed Central

Lundy, Scott D.; Zhu, Wei-Zhong

2013-01-01

Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20–40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80–120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including β-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue. PMID:23461462

The human cumulus--oocyte complex gene-expression profile

PubMed Central

Assou, Said; Anahory, Tal; Pantesco, Véronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Hervé; De Vos, John; Hamamah, Samir

2006-01-01

BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors. PMID:16571642

Artificial Niches for Stromal Stem Cells as a Potential Instrument for the Design of the Surface of Biomimetic Osteogenic Materials

NASA Astrophysics Data System (ADS)

Khlusov, I. A.; Khlusova, M. Yu.; Pichugin, V. F.; Sharkeev, Yu. P.; Legostaeva, E. V.

2014-02-01

A relationship between the topography of rough calcium phosphate surfaces having osteogenic niche-reliefs and the electrostatic potential of these surfaces as a possible instrument to control stromal stem cells has been investigated. The in vitro culture of human lung prenatal stromal cells on nanostructured/ultrafine-grained VT1.0 titanium alloy plates with bilateral rough calcium phosphate (CaP) microarc coating was used. It was established that the amplitude of the electret CaP surface potential linearly increased with increasing area of valleys (sockets), and the negative charge is formed on the socket surface. The area of alkaline phosphatase staining (the marker of osteoblast maturation and differentiation) of adherent CD34- CD44+ cells increases linearly with increasing area of artificial microterritory (socket) of the CaP surface occupied with each cell. The negative electret potential in valleys (sockets) of microarc CaP coatings can be the physical mechanism mediating the influence of the surface topography on osteogenic maturation and differentiation of cells in vitro. This mechanism can be called "niche-potential" and can be used as an instrument for biomimetic modification of smooth CaP surfaces to strengthen their integration with the bone tissue.

Use of Rat Mature Adipocyte-Derived Dedifferentiated Fat Cells as a Cell Source for Periodontal Tissue Regeneration

PubMed Central

Akita, Daisuke; Kano, Koichiro; Saito-Tamura, Yoko; Mashimo, Takayuki; Sato-Shionome, Momoko; Tsurumachi, Niina; Yamanaka, Katsuyuki; Kaneko, Tadashi; Toriumi, Taku; Arai, Yoshinori; Tsukimura, Naoki; Matsumoto, Taro; Ishigami, Tomohiko; Isokawa, Keitaro; Honda, Masaki

2016-01-01

Lipid-free fibroblast-like cells, known as dedifferentiated fat (DFAT) cells, can be generated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and can transdifferentiate into various cell types under appropriate culture conditions. The first objective of this study was to compare the multilineage differentiation potential of DFAT cells with that of adipose-derived stem cells (ASCs) on mesenchymal stem cells. We obtained DFAT cells and ASCs from inbred rats and found that rat DFAT cells possess higher osteogenic differentiation potential than rat ASCs. On the other hand, DFAT cells show similar adipogenic differentiation, and chondrogenic differentiation potential in comparison with ASCs. The second objective of this study was to assess the regenerative potential of DFAT cells combined with novel solid scaffolds composed of PLGA (Poly d, l-lactic-co-glycolic acid) on periodontal tissue, and to compare this with the regenerative potential of ASCs combined with PLGA scaffolds. Cultured DFAT cells and ASCs were seeded onto PLGA scaffolds (DFAT/PLGA and ASCs/PLGA) and transplanted into periodontal fenestration defects in rat mandible. Micro computed tomography analysis revealed a significantly higher amount of bone regeneration in the DFAT/PLGA group compared with that of ASCs/PLGA and PLGA-alone groups at 2, 3, and 5 weeks after transplantation. Similarly, histomorphometric analysis showed that DFAT/PLGA groups had significantly greater width of cementum, periodontal ligament and alveolar bone than ASCs/PLGA and PLGA-alone groups. In addition, transplanted fluorescent-labeled DFAT cells were observed in the periodontal ligament beside the newly formed bone and cementum. These findings suggest that DFAT cells have a greater potential for enhancing periodontal tissue regeneration than ASCs. Therefore, DFAT cells are a promising cell source for periodontium regeneration. PMID:26941649

Rapid and robust generation of long-term self-renewing human neural stem cells with the ability to generate mature astroglia.

PubMed

Palm, Thomas; Bolognin, Silvia; Meiser, Johannes; Nickels, Sarah; Träger, Claudia; Meilenbrock, Ralf-Leslie; Brockhaus, Johannes; Schreitmüller, Miriam; Missler, Markus; Schwamborn, Jens Christian

2015-11-06

Induced pluripotent stem cell bear the potential to differentiate into any desired cell type and hold large promise for disease-in-a-dish cell-modeling approaches. With the latest advances in the field of reprogramming technology, the generation of patient-specific cells has become a standard technology. However, directed and homogenous differentiation of human pluripotent stem cells into desired specific cell types remains an experimental challenge. Here, we report the development of a novel hiPSCs-based protocol enabling the generation of expandable homogenous human neural stem cells (hNSCs) that can be maintained under self-renewing conditions over high passage numbers. Our newly generated hNSCs retained differentiation potential as evidenced by the reliable generation of mature astrocytes that display typical properties as glutamate up-take and expression of aquaporin-4. The hNSC-derived astrocytes showed high activity of pyruvate carboxylase as assessed by stable isotope assisted metabolic profiling. Moreover, using a cell transplantation approach, we showed that grafted hNSCs were not only able to survive but also to differentiate into astroglial in vivo. Engraftments of pluripotent stem cells derived from somatic cells carry an inherent tumor formation potential. Our results demonstrate that hNSCs with self-renewing and differentiation potential may provide a safer alternative strategy, with promising applications especially for neurodegenerative disorders.

What do we know about the participation of hematopoietic stem cells in hematopoiesis?

PubMed

Drize, Nina; Petinati, Nataliya

2015-01-01

The demonstrated presence in adult tissues of cells with sustained tissue regenerative potential has given rise to the concept of tissue stem cells. Assays to detect and measure such cells indicate that they have enormous proliferative potential and usually an ability to produce all or many of the mature cell types that define the specialized functionality of the tissue. In the hematopoietic system, one or only a few cells can restore lifelong hematopoiesis of the whole organism. To what extent is the maintenance of hematopoietic stem cells required during normal hematopoiesis? How does the constant maintenance of hematopoiesis occur and what is the behavior of the hematopoietic stem cells in the normal organism? How many of the hematopoietic stem cells are created during the development of the organism? How many hematopoietic stem cells are generating more mature progeny at any given moment? What happens to the population of hematopoietic stem cells in aging? This review will attempt to describe the results of recent research which contradict some of the ideas established over the past 30 years about how hematopoiesis is regulated.

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

PubMed Central

McElroy, Sohyun L.; Byrne, James A.; Chavez, Shawn L.; Behr, Barry; Hsueh, Aaron J.; Westphal, Lynn M.; Reijo Pera, Renee A.

2010-01-01

Background Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. Methodology/Principal Finding Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. Conclusions/Significance Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer. PMID:20539753

Human periodontal fibroblasts viability stored in Custodiol® , coconut water and propolis. An ex vivo study.

PubMed

Awawdeh, Lama; Haimour, Rana Naman; Al-Jundi, Suhad Hussein; Al-Qaoud, Khaled

2018-04-17

Successful replantation of an avulsed tooth depends on the regeneration of periodontal ligament (PDL) attachment which is affected by the transport medium, dry time and storage time. Various storage media have been studied but the search for the optimum storage medium is still needed to determine the ideal material and storage time to maintain PDL cells. The aim of this study was to determine the ability of Custodiol ® , coconut water from different stages of maturity and propolis as storage media for avulsed teeth by evaluating the viability of PDL cells for different time intervals. PDL cultures were subjected to Cutodiol ® , immature, half mature, and mature coconut water, and different concentrations of propolis in DMEM. Culture plates with the tested media were incubated for 1, 2, 6, 24, 48, 72 and 168 h. PDL fibroblast cell viability was assessed by MTT assay. Coconut water showed significantly higher viability of cells than other groups at 6 h with half mature coconut water being superior. Propolis at 6.25 mg/mL in DMEM resulted in 138% viable PDL and it was able to preserve PDL cells for up to 168 h. Half mature and mature coconut water are superior storage media if replantation of avulsed teeth is within 6 h. Propolis in DMEM could be a potential storage media for prolonged storage intervals up to 48 h. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Microscopic dynamics research on the "mature" process of dye-sensitized solar cells after injection of highly concentrated electrolyte.

PubMed

Liang, Zhongguan; Liu, Weiqing; Chen, Jun; Hu, Linhua; Dai, Songyuan

2015-01-21

After injection of electrolyte, the internal three-dimensional solid-liquid penetration system of dye-sensitized solar cells (DSCs) can take a period of time to reach "mature" state. This paper studies the changes of microscopic processes of DSCs including TiO2 energy-level movement, localized state distribution, charge accumulation, electron transport, and recombination dynamics, from the beginning of electrolyte injection to the time of reached mature state. The results show that the microscopic dynamics process of DSCs exhibited a time-dependent behavior and achieved maturity ∼12 h after injecting the electrolyte into DSCs. Within 0-12 h, several results were observed: (1) the conduction band edge of TiO2 moved slightly toward negative potential direction; (2) the localized states in the band gap of TiO2 was reduced according to the same distribution law; (3) the transport resistance in TiO2 film increased, and electron transport time was prolonged as the time of maturity went on, which indicated that the electron transport process is impeded gradually; (4) the recombination resistance at the TiO2/electrolyte (EL) interface increases, and electron lifetime gradually extends, therefore, the recombination process is continuously suppressed. Furthermore, results suggest that the parameters of EL/Pt-transparent conductive oxide (TCO) interface including the interfacial capacitance, electron-transfer resistance, and transfer time constant would change with time of maturity, indicating that the EL/Pt-TCO interface is a potential factor affecting the mature process of DSCs.

Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues To Regulate Skeletal Muscle Maturation and Vascularization.

PubMed

Tsui, Jonathan H; Janebodin, Kajohnkiart; Ieronimakis, Nicholas; Yama, David M P; Yang, Hee Seok; Chavanachat, Rakchanok; Hays, Aislinn L; Lee, Haeshin; Reyes, Morayma; Kim, Deok-Ho

2017-12-26

Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here, we have developed biomimetic nanopatterned poly(lactic-co-glycolic acid) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles.

Myogenic Maturation by Optical-Training in Cultured Skeletal Muscle Cells.

PubMed

Asano, Toshifumi; Ishizuka, Toru; Yawo, Hiromu

2017-01-01

Optogenetic techniques are powerful tools for manipulating biological processes in identified cells using light under high temporal and spatial resolutions. Here, we describe an optogenetic training strategy to promote morphological maturation and functional development of skeletal muscle cells in vitro. Optical stimulation with a rhythmical frequency facilitates specific structural alignment of sarcomeric proteins. Optical stimulation also depolarizes the membrane potential, and induces contractile responses in synchrony with the given pattern of light pulses. These results suggest that optogenetic techniques can be employed to manipulate activity-dependent processes during myogenic development and control contraction of photosensitive skeletal muscle cells with high temporal and special precision.

Molecular differences between mature and immature dental pulp cells: Bioinformatics and preliminary results.

PubMed

Chen, Long; Jiang, Yifeng; Du, Zhen

2018-04-01

Although previous studies have demonstrated that dental pulp stem cells (DPSCs) from mature and immature teeth exhibit potential for multi-directional differentiation, the molecular and biological difference between the DPSCs from mature and immature permanent teeth has not been fully investigated. In the present study, 500 differentially expressed genes from dental pulp cells (DPCs) in mature and immature permanent teeth were obtained from the Gene Expression Omnibus online database. Based on bioinformatics analysis using the Database for Annotation, Visualization and Integrated Discovery, these genes were divided into a number of subgroups associated with immunity, inflammation and cell signaling. The results of the present study suggest that immune features, response to infection and cell signaling may be different in DPCs from mature and immature permanent teeth; furthermore, DPCs from immature permanent teeth may be more suitable for use in tissue engineering or stem cell therapy. The Online Mendelian Inheritance in Man database stated that Sonic Hedgehog (SHH), a differentially expressed gene in DPCs from mature and immature permanent teeth, serves a crucial role in the development of craniofacial tissues, including teeth, which further confirmed that SHH may cause DPCs from mature and immature permanent teeth to exhibit different biological characteristics. The Search Tool for the Retrieval of Interacting Genes/Proteins database revealed that SHH has functional protein associations with a number of other proteins, including Glioma-associated oncogene (GLI)1, GLI2, growth arrest-specific protein 1, bone morphogenetic protein (BMP)2 and BMP4, in mice and humans. It was also demonstrated that SHH may interact with other genes to regulate the biological characteristics of DPCs. The results of the present study may provide a useful reference basis for selecting suitable DPSCs and molecules for the treatment of these cells to optimize features for tissue engineering or stem cell therapy. Quantitative polymerase chain reaction should be performed to confirm the differential expression of these genes prior to the beginning of a functional study.

Selection of Aptamers for Mature White Adipocytes by Cell SELEX Using Flow Cytometry

PubMed Central

Kim, Eun Young; Kim, Ji Won; Kim, Won Kon; Han, Baek Soo; Park, Sung Goo; Chung, Bong Hyun; Lee, Sang Chul; Bae, Kwang-Hee

2014-01-01

Background Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited. Methods and Results We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes. Conclusions These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity. PMID:24844710

Herbal preparation (HemoHIM) enhanced functional maturation of bone marrow-derived dendritic cells mediated toll-like receptor 4.

PubMed

Lee, Sung-Ju; Kim, Jong-Jin; Kang, Kyung-Yun; Hwang, Yun-Ho; Jeong, Gil-Yeon; Jo, Sung-kee; Jung, Uhee; Park, Hae-Ran; Yee, Sung-Tae

2016-02-19

HemoHIM, which is an herbal preparation of three edible herbs (Angelicam gigas Nakai, Cnidium offinale Makino, and Peaonia japonica Miyabe), is known to have various biological and immunological activities, but the modulatory effects of this preparation on dendritic cells (DCs)-mediated immune responses have not been examined previously. DCs are a unique group of white blood cells that initiate primary immune responses by capturing, processing, and presenting antigens to T cells. In the present study, we investigated the effect of HemoHIM on the functional and phenotypic maturation of murine bone marrow-derived dendritic cells (BMDCs) both in vitro and in vivo. The expression of co-stimulatory molecules (CD40, CD80, CD86, MHC I, and MHC II) and the production of cytokines (IL-1β, IL-6, IL-12p70, and TNF-α) were increased by HemoHIM in BMDCs. Furthermore, the antigen-uptake ability of BMDCs was decreased by HemoHIM, and the antigen-presenting ability of HemoHIM-treated mature BMDCs increased TLR4-dependent CD4(+) and CD8(+) T cell responses. Our findings demonstrated that HemoHIM induces TLR4-mediated BMDCs functional and phenotypic maturation through in vivo and in vitro. And our study showed the antigen-presenting ability that HemoHIM-treated mature BMDCs increase CD4(+) and CD8(+) T cell responses by in vitro. These results suggest that HemoHIM has the potential to mediate DC immune responses.

A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells.

PubMed

Gunhanlar, N; Shpak, G; van der Kroeg, M; Gouty-Colomer, L A; Munshi, S T; Lendemeijer, B; Ghazvini, M; Dupont, C; Hoogendijk, W J G; Gribnau, J; de Vrij, F M S; Kushner, S A

2018-05-01

Progress in elucidating the molecular and cellular pathophysiology of neuropsychiatric disorders has been hindered by the limited availability of living human brain tissue. The emergence of induced pluripotent stem cells (iPSCs) has offered a unique alternative strategy using patient-derived functional neuronal networks. However, methods for reliably generating iPSC-derived neurons with mature electrophysiological characteristics have been difficult to develop. Here, we report a simplified differentiation protocol that yields electrophysiologically mature iPSC-derived cortical lineage neuronal networks without the need for astrocyte co-culture or specialized media. This protocol generates a consistent 60:40 ratio of neurons and astrocytes that arise from a common forebrain neural progenitor. Whole-cell patch-clamp recordings of 114 neurons derived from three independent iPSC lines confirmed their electrophysiological maturity, including resting membrane potential (-58.2±1.0 mV), capacitance (49.1±2.9 pF), action potential (AP) threshold (-50.9±0.5 mV) and AP amplitude (66.5±1.3 mV). Nearly 100% of neurons were capable of firing APs, of which 79% had sustained trains of mature APs with minimal accommodation (peak AP frequency: 11.9±0.5 Hz) and 74% exhibited spontaneous synaptic activity (amplitude, 16.03±0.82 pA; frequency, 1.09±0.17 Hz). We expect this protocol to be of broad applicability for implementing iPSC-based neuronal network models of neuropsychiatric disorders.

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

PubMed Central

van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

2012-01-01

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699

Preosteocytes/Osteocytes Have the Potential to Dedifferentiate Becoming a Source of Osteoblasts

PubMed Central

Torreggiani, Elena; Matthews, Brya G.; Pejda, Slavica; Matic, Igor; Horowitz, Mark C.; Grcevic, Danka; Kalajzic, Ivo

2013-01-01

Presently there is no clear evidence for the ability of mature osteogenic lineage cells to dedifferentiate. In order to identify and trace mature osteogenic lineage cells, we have utilized transgenic mouse models in which the dentin matrix protein 1 (Dmp1) promoter drives expression of GFP (active marker) or Cre recombinase (historic label) in preosteocytes/osteocytes. In long bone chip outgrowth cultures, in which cells on the bone surface were enzymatically removed, cells with previous activity of the Dmp1 promoter migrated onto plastic and down-regulated Dmp1-GFP expression. Dmp1Cre-labeled cells from these cultures had the potential to re-differentiate into the osteogenic lineage, while the negative population showed evidence of adipogenesis. We observed numerous Dmp1Cre-labeled osteoblasts on the surface of bone chips following their in vivo transplantation. Our data indicate that cells embedded in bone matrix are motile, and once given access to the extra bony milieu will migrate out of their lacunae. This population of cells is phenotypically and functionally heterogeneous in vitro. Once the preosteocytes/osteocytes leave lacunae, they can dedifferentiate, potentially providing an additional source of functional osteoblasts. PMID:24040401

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection.

PubMed

Magarey, Genevieve M; Mate, Karen E

2004-01-01

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte(-1) h(-1)), in vitro- matured (0.93 pmol oocyte(-1) h(-1)) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte(-1) h(-1)) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17-19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

PubMed Central

Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

2013-01-01

Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID:24349574

Engineering stem cells for future medicine.

PubMed

Ricotti, Leonardo; Menciassi, Arianna

2013-03-01

Despite their great potential in regenerative medicine applications, stem cells (especially pluripotent ones) currently show a limited clinical success, partly due to a lack of biological knowledge, but also due to a lack of specific and advanced technological instruments able to overcome the current boundaries of stem cell functional maturation and safe/effective therapeutic delivery. This paper aims at describing recent insights, current limitations, and future horizons related to therapeutic stem cells, by analyzing the potential of different bioengineering disciplines in bringing stem cells toward a safe clinical use. First, we clarify how and why stem cells should be properly engineered and which could be in a near future the challenges and the benefits connected with this process. Second, we identify different routes toward stem cell differentiation and functional maturation, relying on chemical, mechanical, topographical, and direct/indirect physical stimulation. Third, we highlight how multiscale modeling could strongly support and optimize stem cell engineering. Finally, we focus on future robotic tools that could provide an added value to the extent of translating basic biological knowledge into clinical applications, by developing ad hoc enabling technologies for stem cell delivery and control.

Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

NASA Technical Reports Server (NTRS)

Chakravarthy, M. V.; Abraha, T. W.; Schwartz, R. J.; Fiorotto, M. L.; Booth, F. W.

2000-01-01

Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

Construction of an Expression System for Bioactive IL-18 and Generation of Recombinant Canine Distemper Virus Expressing IL-18

PubMed Central

LIU, Yuxiu; SATO, Hiroki; HAMANA, Masahiro; MOONAN, Navita Anisia; YONEDA, Misako; XIA, Xianzhu; KAI, Chieko

2014-01-01

ABSTRACT Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo. PMID:24898077

Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

PubMed

Liu, Yuxiu; Sato, Hiroki; Hamana, Masahiro; Moonan, Navita Anisia; Yoneda, Misako; Xia, Xianzhu; Kai, Chieko

2014-09-01

Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

Stem Cell, Regenerative Medicine and Cancer | Center for Cancer Research

Cancer.gov

Of the estimated trillion cells that build up our bodies, only a little number can self-renew and give rise to many different cell types. These unspecialized cells are called stem cells. Stem cell division and differentiation is fundamental to the development of the mature organism. Stem cells have recently attracted significant attention largely due to their potential medical

Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons.

PubMed

Broccoli, Vania; Rubio, Alicia; Taverna, Stefano; Yekhlef, Latefa

2015-06-01

The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons. © 2015 by the Society for Experimental Biology and Medicine.

Dynamic pressurization induces transition of notochordal cells to a mature phenotype while retaining production of important patterning ligands from development

PubMed Central

2013-01-01

Introduction Notochordal cells (NCs) pattern aneural and avascular intervertebral discs (IVDs), and their disappearance, is associated with onset of IVD degeneration. This study induced and characterized the maturation of nucleus pulposus (NP) tissue from a gelatinous NC-rich structure to a matrix-rich structure populated by small NP cells using dynamic pressurization in an ex vivo culture model, and also identified soluble factors from NCs with therapeutic potential. Methods Porcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5 to 2 MPa at 0.1 Hz for 2 hours) either Daily, for 1 Dose, or Control (no pressurization) groups for up to eight days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics. Results Daily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for Control, 1 Dose and Daily groups, respectively (P < 0.0002) and no relevant cell death. Dynamic loading matured NP tissue by significantly increasing metabolic activity and accumulating Safranin-O-stained matrix. Load-induced maturation was also apparent from the significantly decreased glycolytic, cytoskeletal (Vimentin) and stress-inducible (HSP70) proteins assessed with proteomics. Loading increased the production of bioactive proteins Sonic Hedgehog (SHH) and Noggin, and maintained Semaphorin3A (Sema3A). Discussion NP tissue maturation was induced from dynamic hydrostatic pressurization in a controlled ex vivo environment without influence from systemic effects or surrounding structures. NCs transitioned into small nonvacuolated NP cells probably via differentiation as evidenced by high cell viability, lack of nitric oxide and downregulation of stress-inducible and cytoskeletal proteins. SHH, Sema3A, and Noggin, which have patterning and neurovascular-inhibiting properties, were produced in both notochordal and matured porcine NP. Results therefore provide an important piece of evidence suggesting the transition of NCs to small NP cells is a natural part of aging and not the initiation of degeneration. Bioactive candidates identified from young porcine IVDs may be isolated and harnessed for therapies to target discogenic back pain. PMID:24427812

Dynamic pressurization induces transition of notochordal cells to a mature phenotype while retaining production of important patterning ligands from development.

PubMed

Purmessur, Devina; Guterl, Clare C; Cho, Samuel K; Cornejo, Marisa C; Lam, Ying W; Ballif, Bryan A; Laudier, James C Iatridis; Iatridis, James C

2013-01-01

Notochordal cells (NCs) pattern aneural and avascular intervertebral discs (IVDs), and their disappearance, is associated with onset of IVD degeneration. This study induced and characterized the maturation of nucleus pulposus (NP) tissue from a gelatinous NC-rich structure to a matrix-rich structure populated by small NP cells using dynamic pressurization in an ex vivo culture model, and also identified soluble factors from NCs with therapeutic potential. Porcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5 to 2 MPa at 0.1 Hz for 2 hours) either Daily, for 1 Dose, or Control (no pressurization) groups for up to eight days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics. Daily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for Control, 1 Dose and Daily groups, respectively (P < 0.0002) and no relevant cell death. Dynamic loading matured NP tissue by significantly increasing metabolic activity and accumulating Safranin-O-stained matrix. Load-induced maturation was also apparent from the significantly decreased glycolytic, cytoskeletal (Vimentin) and stress-inducible (HSP70) proteins assessed with proteomics. Loading increased the production of bioactive proteins Sonic Hedgehog (SHH) and Noggin, and maintained Semaphorin3A (Sema3A). NP tissue maturation was induced from dynamic hydrostatic pressurization in a controlled ex vivo environment without influence from systemic effects or surrounding structures. NCs transitioned into small nonvacuolated NP cells probably via differentiation as evidenced by high cell viability, lack of nitric oxide and downregulation of stress-inducible and cytoskeletal proteins. SHH, Sema3A, and Noggin, which have patterning and neurovascular-inhibiting properties, were produced in both notochordal and matured porcine NP. Results therefore provide an important piece of evidence suggesting the transition of NCs to small NP cells is a natural part of aging and not the initiation of degeneration. Bioactive candidates identified from young porcine IVDs may be isolated and harnessed for therapies to target discogenic back pain.

Activity-Dependent Changes in the Firing Properties of Neocortical Fast-Spiking Interneurons in the Absence of Large Changes in Gene Expression

PubMed Central

Miller, Mark N.; Okaty, Benjamin W.; Kato, Saori; Nelson, Sacha B.

2010-01-01

The diverse cell types that comprise neocortical circuits each have characteristic integrative and firing properties that are specialized to perform specific functions within the network. Parvalbumin-positive fast-spiking (FS) interneurons are a particularly specialized cortical cell-type that controls the dynamics of ongoing activity and prevents runaway excitation by virtue of remarkably high firing rates, a feature that is permitted by narrow action potentials and the absence of spike-frequency adaptation. Although several neuronal intrinsic membrane properties undergo activity-dependent plasticity, the role of network activity in shaping and maintaining specialized, cell-type-specific firing properties is unknown. We tested whether the specialized firing properties of mature FS interneurons are sensitive to activity perturbations by inactivating a portion of motor cortex in vivo for 48 hours and measuring resulting plasticity of FS intrinsic and firing properties with whole-cell recording in acute slices. Many of the characteristic properties of FS interneurons, including non-adapting high-frequency spiking and narrow action potentials, were profoundly affected by activity deprivation both at an age just after maturation of FS firing properties and also a week after their maturation. Using microarray screening, we determined that although normal maturation of FS electrophysiological specializations is accompanied by large-scale transcriptional changes, the effects of deprivation on the same specializations involve more modest transcriptional changes, and may instead be primarily mediated by post-transcriptional mechanisms. PMID:21154910

Development of the terminally differentiated state sensitizes epiphyseal chondrocytes to apoptosis through caspase-3 activation.

PubMed

Pucci, Bruna; Adams, Christopher S; Fertala, Jolanta; Snyder, Bradley C; Mansfield, Kyle D; Tafani, Marco; Freeman, Theresa; Shapiro, Irving M

2007-03-01

The maturation of epiphyseal chondrocytes is accompanied by dramatic changes in energy metabolism and shifts in proteins concerned with the induction of apoptosis. We evaluated the role of mitochondria in this process by evaluating the membrane potential (Delta psi m) of chondrocytes of embryonic tibia and the epiphyseal growth plate. We observed that there was a maturation-dependent change in fluorescence, indicating a fall in the Delta psi m. The level of mitochondrial Bcl-2 was decreased during maturation, while in the same time period there was an obvious increase in Bax levels in the mitochondrial fraction of the terminally differentiated chondrocytes. Bcl(xL), another anti-apoptotic protein, was also robustly expressed in the mitochondrial fraction, but its expression was not dependent on the maturation status of the chondrocytes. We found that caspase-3 was present throughout the growth plate and in hypertrophic cells in culture. We blocked caspase-3 activity and found that alkaline phosphatase staining and mineral formation was decreased, and the cells had lost their characteristic shape. Moreover, we noted that the undifferentiated cells were insensitive to elevated concentrations of inorganic phosphate (Pi). It is concluded that during hypertrophy, the change in membrane potential, the increased binding of a pro-apoptotic protein to mitochondria, and the activation of caspase-3 serve to prime cells for apoptosis. Only when the terminally differentiated chondrocytes are challenged with low levels of apoptogens there is activation of apoptosis. Copyright 2006 Wiley-Liss, Inc.

Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Negoro, Ryosuke; Takayama, Kazuo; The Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research

Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cellsmore » were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. - Highlights: • The hiPS-ELCs were matured by Matrigel overlay. • The hiPS-ELCs expressed intestinal nuclear receptors, such as PXR, GR and VDR. • The hiPS-ELC is a useful model for the drug-mediated CYP3A4 induction test.« less

Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network.

PubMed

Perdigoto, Carolina N; Bardot, Evan S; Valdes, Victor J; Santoriello, Francis J; Ezhkova, Elena

2014-12-01

Merkel cell-neurite complexes are located in touch-sensitive areas of the mammalian skin and are involved in recognition of the texture and shape of objects. Merkel cells are essential for these tactile discriminations, as they generate action potentials in response to touch stimuli and induce the firing of innervating afferent nerves. It has been shown that Merkel cells originate from epidermal stem cells, but the cellular and molecular mechanisms of their development are largely unknown. In this study, we analyzed Merkel cell differentiation during development and found that it is a temporally regulated maturation process characterized by a sequential activation of Merkel cell-specific genes. We uncovered key transcription factors controlling this process and showed that the transcription factor Atoh1 is required for initial Merkel cell specification. The subsequent maturation steps of Merkel cell differentiation are controlled by cooperative function of the transcription factors Sox2 and Isl1, which physically interact and work to sustain Atoh1 expression. These findings reveal the presence of a robust transcriptional network required to produce functional Merkel cells that are required for tactile discrimination. © 2014. Published by The Company of Biologists Ltd.

Induction of human dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4.

PubMed

Cisco, Robin M; Abdel-Wahab, Zeinab; Dannull, Jens; Nair, Smita; Tyler, Douglas S; Gilboa, Eli; Vieweg, Johannes; Daaka, Yehia; Pruitt, Scott K

2004-06-01

Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

Development of immature stomata: evidence for epigenetic selection of a spacing pattern.

PubMed

Kagan, M L; Sachs, T

1991-07-01

In Sansevieria trifasciata as many as half the potential stomata remain immature. The development of all stomatal structures started at the same time and the early stages of the development of immature stomata had no special characteristics. Statistical analysis showed that the mature stomata were more evenly spaced than all potential stomata, both mature and immature. Furthermore, the distribution of mature stomata per unit area was more predictable or orderly than comparable structures of a random model that developed in the same way. These facts indicate that a nonrandom loss of many stomata by "immaturity" is a major determinant, acting during rather than preceding development, of the distribution of the mature, functional stomata. Thus in Sansevieria there is a selection of an epidermal pattern from an excess of cells that undergo the early stages of stomatal development.

Saquinavir inhibits early events associated with establishment of HIV-1 infection: potential role for protease inhibitors in prevention.

PubMed

Stefanidou, Martha; Herrera, Carolina; Armanasco, Naomi; Shattock, Robin J

2012-08-01

The maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency against trans infection of T cells by in vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.

Somatic and germinal cells' interrelationship in the course of seminiferous tubule maturation in man.

PubMed

Kula, K; Romer, T E; Wlodarczyk, W P

1980-02-01

Certain successive phases of seminiferous tubule maturation were observed in a transsection of a Leydig cell adenoma-bearing testis of a boy with precocious puberty. Massively accumulated Leydig cells may stimulate the maturation of Sertoli cells, as indicated by progressive replacement of Sertoli cell precursors by mature Sertoli cells at a distance closer to the adenoma. On the other hand, tubules less advanced in maturation contained a higher number of somatic cells than those more advanced in maturation. Leydig-cell-dependent maturation of Sertoli cells may be in competition with Certoli cell multiplication, or numerous undifferentiated somatic cells may undergo a natural elimination in the course of tubular maturation. An inverse relation between the number of Sertoli cell precursors and the number of meiotic spermatocytes suggests that quantitative reduction of Sertoli cell precursors may be important for the intratubular milieu necessary for the onset of the first meiosis in man.

Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

NASA Technical Reports Server (NTRS)

Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

2000-01-01

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms

PubMed Central

Jackson, Desmond N.; Yang, Lin; Wu, ShiBiao; Kennelly, Edward J.

2015-01-01

Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 μM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC50s) of 30 to 50 μM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC50 from >1,024 μg/ml to 13 μg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis. PMID:26195512

Contrasting effects of TGF-beta 1 and TNF-alpha on the development of dendritic cells from progenitors in mouse bone marrow.

PubMed

Yamaguchi, Y; Tsumura, H; Miwa, M; Inaba, K

1997-01-01

Dendritic cells (DC) are a distinct population of leukocytes and specialized antigen-presenting cells for T cell responses. Prior work has shown that GM-CSF can induce the development of large numbers of DC from proliferating progenitors in mouse bone marrow. We have monitored the effects of potentially enhancing and suppressive cytokines in these cultures. In this system, many immature DC develop from proliferating precursors during the first six days of culture, and between days 6-8 maturation of typical nonadherent and nonreplicating DC takes place. The maturation is accompanied by a large increase in the expression of major histocompatibilities complex class II (MHC II) and B7-2/CD86, and in mixed leukocyte reaction stimulating activity. Tumor necrosis factor-alpha (TNF-alpha), previously shown to be required for development of human DC, was found to enhance the maturation of mouse DC in the last two days of culture. Transforming growth factor-beta 1 (TGF-beta 1), on the other hand, almost totally blocked DC maturation, but it had to be given in the first six days of culture when the DC were actively proliferating. TGF-beta 1 did not block the production of immature, MHC II-positive but B7-2/CD86-negative DC. Maturation would take place between days 6-8 as long as the cultures were depleted of Fc-receptor-bearing cells, or if TNF-alpha were added. In both instances, maturation was not blocked even when TGF-beta 1 remained in the culture. We conclude that the development of DC, in response to GM-CSF, can be modified by other cytokines. TGF-beta 1 is suppressive but only indirectly via Fc-receptor-bearing suppressive cells, presumably suppressive macrophages, while TNF-alpha enhances the final maturation of DC.

Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations

PubMed Central

Griffin, Síle M.; Pickard, Mark R.; Orme, Rowan P.; Hawkins, Clive P.; Williams, Adrian C.

2017-01-01

Introduction Vitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington’s disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons. Methods Nicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods. Results Results presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons. Conclusions Our results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons. PMID:28817722

Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure

PubMed Central

Mohan, Joanne; Hopkins, John; Mabbott, Neil A

2005-01-01

The accumulation of the scrapie agent in lymphoid tissues following inoculation via the skin is critical for efficient neuroinvasion, but how the agent is initially transported from the skin to the draining lymph node is not known. Langerhans cells (LCs) are specialized antigen-presenting cells that continually sample their microenvironment within the epidermis and transport captured antigens to draining lymph nodes. We considered LCs probable candidates to acquire and transport the scrapie agent after inoculation via the skin. XS106 cells are dendritic cells (DCs) isolated from mouse epidermis with characteristics of mature LC cells. To investigate the potential interaction of LCs with the scrapie agent XS106 cells were exposed to the scrapie agent in vitro. We show that XS106 cells rapidly acquire the scrapie agent following in vitro exposure. In addition, XS106 cells partially degrade the scrapie agent following extended cultivation. These data suggest that LCs might acquire and degrade the scrapie agent after inoculation via the skin, but data from additional experiments demonstrate that this ability could be lost in the presence of lipopolysaccharide or other immunostimulatory molecules. Our studies also imply that LCs would not undergo maturation following uptake of the scrapie agent in the skin, as the expression of surface antigens associated with LC maturation were unaltered following exposure. In conclusion, although LCs or DCs have the potential to acquire the scrapie agent within the epidermis our data suggest it is unlikely that they become activated and stimulated to transport the agent to the draining lymph node. PMID:16108824

Norm- and hypo-fractionated radiotherapy is capable of activating human dendritic cells.

PubMed

Kulzer, Lorenz; Rubner, Yvonne; Deloch, Lisa; Allgäuer, Andrea; Frey, Benjamin; Fietkau, Rainer; Dörrie, Jan; Schaft, Niels; Gaipl, Udo S

2014-10-01

Despite the transient immunosuppressive properties of local radiotherapy (RT), this classical treatment modality of solid tumors is capable of inducing immunostimulatory forms of tumor-cell death. The resulting 'immunotoxicity' in the tumor, but not in healthy tissues, may finally lead to immune-mediated destruction of the tumor. However, little is known about the best irradiation scheme in this setting. This study examines the immunological effects of differently irradiated human colorectal tumor cells on human monocyte-derived dendritic cells (DC). Human SW480 tumor cells were irradiated with a norm-fractionation scheme (5 × 2 Gy), a hypo-fractionated protocol (3 × 5 Gy), and with a high single irradiation dose (radiosurgery; 1 × 15 Gy). Subsequently, human immature DC (iDC) were co-incubated with supernatants (SN) of these differently treated tumor cells. Afterwards, DC were analyzed regarding the expression of maturation markers, the release of cytokines, and the potential to stimulate CD4(+) T-cells. The co-incubation of iDC with SN of tumor cells exposed to norm- or hypo-fractionated RT resulted in a significantly increased secretion of the immune activating cytokines IL-12p70, IL-8, IL-6, and TNFα, compared to iDC co-incubated with SN of tumor cells that received a high single irradiation dose or were not irradiated. In addition, DC-maturation markers CD80, CD83, and CD25 were also exclusively elevated after co-incubation with the SN of fractionated irradiated tumor cells. Furthermore, the SN of tumor cells that were irradiated with norm- or hypo-fractionated RT triggered iDC to stimulate CD4(+) T-cells not only in an allogenic, but also in an antigen-specific manner like mature DC. Collectively, these results demonstrate that norm- and hypo-fractionated RT induces a fast human colorectal tumor-cell death with immunogenic potential that can trigger DC maturation and activation in vitro. Such findings may contribute to the improvement of irradiation protocols for the most beneficial induction of anti-tumor immunity.

Different protein of Echinococcus granulosus stimulates dendritic induced immune response.

PubMed

Wang, Yana; Wang, Qiang; Lv, Shiyu; Zhang, Shengxiang

2015-06-01

Cystic echinococcosis is a chronic infectious disease that results from a host/parasite interaction. Vaccination with ferritin derived from Echinococcus granulosus is a potential preventative treatment. To understand whether ferritin is capable of inducing a host immune response, we investigated the response of dendritic cells (DCs) to both recombinant ferritin protein and the hydatid fluid (HF) of E. granulosus. We evaluated the immunomodulatory potential of these antigens by performing, immunocytochemistry, electron microscopy and in vivo imaging of monocyte-derived murine DCs. During antigen stimulation of DCs, ferritin cause DCs maturation and induced higher levels of surface marker expression and activated T-cell proliferation and migration. On contrary, HF failed to induce surface marker expression and to stimulate T-cell proliferation. In response to HF, DCs produced interleukin-6 (IL-6), but no IL-12 and IL-10. DCs stimulated with ferritin produced high levels of cytokines. Overall, HF appears to induce host immunosuppression in order to ensure parasite survival via inhibits DC maturation and promotes Th2-dependent secretion of cytokines. Although ferritin also promoted DC maturation and cytokine release, it also activates CD4+T-cell proliferation, but regard of the mechanism of the Eg.ferritin induce host to eradicate E. granulosus were not clear.

Predicting the influence of liposomal lipid composition on liposome size, zeta potential and liposome-induced dendritic cell maturation using a design of experiments approach.

PubMed

Soema, Peter C; Willems, Geert-Jan; Jiskoot, Wim; Amorij, Jean-Pierre; Kersten, Gideon F

2015-08-01

In this study, the effect of liposomal lipid composition on the physicochemical characteristics and adjuvanticity of liposomes was investigated. Using a design of experiments (DoE) approach, peptide-containing liposomes containing various lipids (EPC, DOPE, DOTAP and DC-Chol) and peptide concentrations were formulated. Liposome size and zeta potential were determined for each formulation. Moreover, the adjuvanticity of the liposomes was assessed in an in vitro dendritic cell (DC) model, by quantifying the expression of DC maturation markers CD40, CD80, CD83 and CD86. The acquired data of these liposome characteristics were successfully fitted with regression models, and response contour plots were generated for each response factor. These models were applied to predict a lipid composition that resulted in a liposome with a target zeta potential. Subsequently, the expression of the DC maturation factors for this lipid composition was predicted and tested in vitro; the acquired maturation responses corresponded well with the predicted ones. These results show that a DoE approach can be used to screen various lipids and lipid compositions, and to predict their impact on liposome size, charge and adjuvanticity. Using such an approach may accelerate the formulation development of liposomal vaccine adjuvants. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Direct Demonstration of a Growth-Induced Water Potential Gradient.

PubMed

Nonami, H.; Boyer, J. S.

1993-05-01

When transpiration is negligible, water potentials in growing tissues are less than those in mature tissues and have been predicted to form gradients that move water into the enlarging cells. To determine directly whether the gradients exist, we measured water potentials along the radius of stems of intact soybean (Glycine max [L.] Merr.) seedlings growing in vermiculite in a water-saturated atmosphere. The measurements were made in individual cells by first determining the turgor with a miniature pressure probe, then determining the osmotic potential of solution from the same cell, and finally summing the two potentials. The osmotic potentials were corrected for sample mixing in the probe. The measurements were checked with a thermocouple psychrometer that gave average tissue water potentials. In the elongating region, the water potential was highest near the xylem and lowest near the epidermis and in the center of the pith. In the basal, more mature region of the same stems, water potentials were near zero next to the xylem and throughout the tissue. These basal potentials reflected mostly the potential of the xylem, which extended into the elongating tissues. Thus, the high basal potential confirmed the high potential near the xylem in the elongating tissues. The psychrometer measurements for each tissue gave average potentials that agreed with the average of the cell potentials from the pressure probe. We conclude that a radial gradient was present in the elongating region that formed a water potential field in three dimensions around the xylem and that confirmed the predictions of Molz and Boyer (F.J. Molz and J.S. Boyer [1978] Plant Physiol 62: 423-429).

Direct Demonstration of a Growth-Induced Water Potential Gradient.

PubMed Central

Nonami, H.; Boyer, J. S.

1993-01-01

When transpiration is negligible, water potentials in growing tissues are less than those in mature tissues and have been predicted to form gradients that move water into the enlarging cells. To determine directly whether the gradients exist, we measured water potentials along the radius of stems of intact soybean (Glycine max [L.] Merr.) seedlings growing in vermiculite in a water-saturated atmosphere. The measurements were made in individual cells by first determining the turgor with a miniature pressure probe, then determining the osmotic potential of solution from the same cell, and finally summing the two potentials. The osmotic potentials were corrected for sample mixing in the probe. The measurements were checked with a thermocouple psychrometer that gave average tissue water potentials. In the elongating region, the water potential was highest near the xylem and lowest near the epidermis and in the center of the pith. In the basal, more mature region of the same stems, water potentials were near zero next to the xylem and throughout the tissue. These basal potentials reflected mostly the potential of the xylem, which extended into the elongating tissues. Thus, the high basal potential confirmed the high potential near the xylem in the elongating tissues. The psychrometer measurements for each tissue gave average potentials that agreed with the average of the cell potentials from the pressure probe. We conclude that a radial gradient was present in the elongating region that formed a water potential field in three dimensions around the xylem and that confirmed the predictions of Molz and Boyer (F.J. Molz and J.S. Boyer [1978] Plant Physiol 62: 423-429). PMID:12231794

Yttrium Y 90 Basiliximab and Combination Chemotherapy Before Stem Cell Transplant in Treating Patients With Mature T-cell Non-Hodgkin Lymphoma

ClinicalTrials.gov

2018-04-10

Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Recurrent Mature T- and NK-Cell Non-Hodgkin Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Recurrent Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory Cutaneous T-Cell Non-Hodgkin Lymphoma

IK1-enhanced human-induced pluripotent stem cell-derived cardiomyocytes: an improved cardiomyocyte model to investigate inherited arrhythmia syndromes

PubMed Central

Vaidyanathan, Ravi; Markandeya, Yogananda S.; Kamp, Timothy J.; Makielski, Jonathan C.; January, Craig T.

2016-01-01

Currently available induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) do not ideally model cellular mechanisms of human arrhythmic disease due to lack of a mature action potential (AP) phenotype. In this study, we create and characterize iPS-CMs with an electrically mature AP induced by potassium inward rectifier (IK1) enhancement. The advantages of IK1-enhanced iPS-CMs include the absence of spontaneous beating, stable resting membrane potentials at approximately −80 mV and capability for electrical pacing. Compared with unenhanced, IK1-enhanced iPS-CMs calcium transient amplitudes were larger (P < 0.05) with a typical staircase pattern. IK1-enhanced iPS-CMs demonstrated a twofold increase in cell size and membrane capacitance and increased DNA synthesis compared with control iPS-CMs (P < 0.05). Furthermore, IK1-enhanced iPS-CMs expressing the F97C-CAV3 long QT9 mutation compared with wild-type CAV3 demonstrated an increase in AP duration and late sodium current. IK1-enhanced iPS-CMs represent a more mature cardiomyocyte model to study arrhythmia mechanisms. PMID:27059077

Characterization of CD34+ hematopoietic cells in systemic mastocytosis: Potential role in disease dissemination.

PubMed

Mayado, A; Teodosio, C; Dasilva-Freire, N; Jara-Acevedo, M; Garcia-Montero, A C; Álvarez-Twose, I; Sánchez-Muñoz, L; Matito, A; Caldas, C; Muñoz-González, J I; Henriques, A; Sánchez-Gallego, J I; Escribano, L; Orfao, A

2018-01-13

Recent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs-), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation-associated compartments of bone marrow (BM) and peripheral blood (PB) CD34 + hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation. The distribution of different maturation-associated subsets of BM and PB CD34 + HPC from 64 newly diagnosed (KIT-mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS-purified PB cell compartments were also investigated for the KIT mutation. ISM patients showed higher percentages of both BM and PB MC-committed CD34 + HPC vs controls, particularly among ISM cases with MC-restricted KIT mutation (ISM MC ); this was associated with progressive blockade of maturation of CD34 + HPC to the neutrophil lineage from ISM MC to multilineage KIT-mutated cases (ISM ML ). Regarding the frequency of KIT-mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT-mutated PB CD34 + HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs- MC and ISMs+ MC to ISM ML patients. The presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34 + HPC and multiple myeloid cell subpopulations, KIT-mutated PB CD34 + HPC potentially contributing to early dissemination of the disease. © 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Rapid integration of young newborn dentate gyrus granule cells in the adult hippocampal circuitry.

PubMed

Ide, Yoko; Fujiyama, Fumino; Okamoto-Furuta, Keiko; Tamamaki, Nobuaki; Kaneko, Takeshi; Hisatsune, Tatsuhiro

2008-12-01

Newborn dentate gyrus granule cells (DGCs) are integrated into the hippocampal circuitry and contribute to the cognitive functions of learning and memory. The dendritic maturation of newborn DGCs in adult mice occurs by the first 3-4 weeks, but DGCs seem to receive a variety of neural inputs at both their dendrites and soma even shortly after their birth. However, few studies on the axonal maturation of newborn DGCs have focused on synaptic structure. Here, we investigated the potentiality of output and input in newborn DGCs, especially in the early period after terminal mitosis. We labeled nestin-positive progenitor cells by injecting GFP Cre-reporter adenovirus into Nestin-Cre mice, enabling us to trace the development of progenitor cells by their GFP expression. In addition to GABAergic input from interneurons, we observed that the young DGCs received axosomatic input from the medial septum as early as postinfection day 7 (PID 7). To evaluate the axonal maturation of the newborn DGCs compared with mature DCGs, we performed confocal and electron microscopic analyses. We observed that newborn DGCs projected their mossy fibers to the CA3 region, forming small terminals on hilar or CA3 interneurons and large boutons on CA3 pyramidal cells. These terminals expressed vesicular glutamate transporter 1, indicating they were glutamatergic terminals. Intriguingly, the terminals at PID 7 had already formed asymmetric synapses, similar to those of mature DGCs. Together, our findings suggest that newborn DGCs may form excitatory synapses on both interneurons and CA3 pyramidal cells within 7 days of their terminal mitosis.

Estrogen alters gonadal soma-derived factor (Gsdf)/Foxl2 expression levels in the testes associated with testis-ova differentiation in adult medaka, Oryzias latipes.

PubMed

Kobayashi, Tohru; Chiba, Ayaka; Sato, Tadashi; Myosho, Taijun; Yamamoto, Jun; Okamura, Tetsuro; Onishi, Yuta; Sakaizumi, Mitsuru; Hamaguchi, Satoshi; Iguchi, Taisen; Horie, Yoshifumi

2017-10-01

Testis-ova differentiation in sexually mature male medaka (Oryzias latipes) is easily induced by estrogenic chemicals, indicating that spermatogonia persist in sexual bipotentiality, even in mature testes in medaka. By contrast, the effects of estrogen on testicular somatic cells associated with testis-ova differentiation in medaka remain unclear. In this study, we focused on the dynamics of sex-related genes (Gsdf, Dmrt1, and Foxl2) expressed in Sertoli cells in the mature testes of adult medaka during estrogen-induced testis-ova differentiation. When mature male medaka were exposed to estradiol benzoate (EB; 800ng/L), testis-ova first appeared after EB treatment for 14days (observed as the first oocytes of the leptotene-zygotene stage). However, the testis remained structurally unchanged, even after EB treatment for 28days. Although Foxl2 is a female-specific sex gene, EB treatment for 7days induced Foxl2/FOXL2 expression in all Sertoli cell-enclosed spermatogonia before testis-ova first appeared; however, Foxl2 was not detected in somatic cells in control testes. Conversely, Sertoli-cell-specific Gsdf mRNA expression levels significantly decreased after EB treatment for 14days, and no changes were observed in DMRT1 localization following EB treatment, whereas Dmrt1 mRNA levels increased significantly. Furthermore, after EB exposure, FOXl2 and DMRT1 were co-localized in Sertoli cells during testis-ova differentiation, although FOXL2 localization was undetectable in Sertoli-cell-enclosed apoptotic testis-ova, whereas DMRT1 remained localized in Sertoli cells. These results indicated for the first time that based on the expression of female-specific sex genes, feminization of Sertoli cells precedes testis-ova differentiation induced by estrogen in mature testes in medaka; however, complete feminization of Sertoli cells was not induced in this study. Additionally, it is suggested strongly that Foxl2 and Gsdf expression constitute potential molecular markers for evaluating the effects of estrogenic chemicals on testicular somatic cells associated with estrogen-induced testis-ova differentiation in mature male medaka. Copyright © 2017 Elsevier B.V. All rights reserved.

Red blood cell generation by three-dimensional aggregate cultivation of late erythroblasts.

PubMed

Lee, EunMi; Han, So Yeon; Choi, Hye Sook; Chun, Bokhwan; Hwang, Byunghee; Baek, Eun Jung

2015-02-01

Stem cell-derived erythroid cells hold great potential for the treatment of blood-loss anemia and for erythropoiesis research; however, cultures using conventional flat plates or bioreactors have failed to show promising results. By mimicking the in vivo bone marrow (BM) environment in which most erythroid cells are physically aggregated, we show that a three-dimensional (3D) aggregate culture system facilitates erythroid cell maturation and red blood cell (RBC) production more effectively than two-dimensional high-density cell cultivation. Late erythroblasts (polychromatic or orthochromatic erythroblasts) were differentiated from cord blood CD34(+) cells over 15 days and then allowed to form tight aggregates at a minimum density of 1×10(7) cells/mL for 2-3 days. To scale up the cell culture and to make the media supply efficient throughout the cell aggregates, several macroporous microcarriers and porous scaffolds were applied to the 3D culture system. In comparison to control culture conditions, erythroid cells in 3D aggregates were significantly more differentiated toward RBCs with significantly reduced nuclear dysplasia. When 3D culture was performed inside macroporous microcarriers, the cell culture scale was increased and cells exhibited enhanced differentiation and enucleation. Microcarriers with a pore diameter of approximately 400 μm produced more mature cells than those with a smaller pore diameter. In addition, this aggregate culture method minimized the culture space and media volume required. In conclusion, a 3D aggregate culture system can be used to generate transfusable human erythrocytes at the terminal maturation stage, mimicking the in vivo BM microenvironment. Porous structures can efficiently maximize the culture scale, enabling large-scale production of RBCs. These results enhance our understanding of the importance of physical contact among late erythroblasts for their final maturation into RBCs.

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration.

PubMed

Van de Walle, Aurore; Wilhelm, Claire; Luciani, Nathalie

2017-04-27

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Efficient Direct Reprogramming of Mature Amniotic Cells into Endothelial Cells by ETS Factors and TGFβ Suppression

PubMed Central

Ginsberg, Michael; James, Daylon; Ding, Bi-Sen; Nolan, Daniel; Geng, Fuqiang; Butler, Jason M; Schachterle, William; Pulijaal, Venkat R; Mathew, Susan; Chasen, Stephen T; Xiang, Jenny; Rosenwaks, Zev; Shido, Koji; Elemento, Olivier; Rabbany, Sina Y; Rafii, Shahin

2012-01-01

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders. PMID:23084400

Cervical (pre)neoplastic microenvironment promotes the emergence of tolerogenic dendritic cells via RANKL secretion

PubMed Central

Demoulin, Stéphanie A; Somja, Joan; Duray, Anaëlle; Guénin, Samuel; Roncarati, Patrick; Delvenne, Philippe O; Herfs, Michael F; Hubert, Pascale M

2015-01-01

The progression of genital human papillomavirus (HPV) infections into preneoplastic lesions suggests that infected/malignant cells are not adequately recognized by the immune system. In this study, we demonstrated that cervical/vulvar cancer cells secrete factor(s) that affect both the maturation and function of dendritic cells (DC) leading to a tolerogenic profile. Indeed, DC cocultured with cancer cell lines display both a partially mature phenotype after lipopolysaccharide (LPS) maturation and an altered secretory profile (IL-10high and IL-12p70low). In addition, tumor-converted DC acquire the ability to alter T-cell proliferation and to induce FoxP3+ suppressive T cells from naive CD4+ T cells. Among the immunosuppressive factors implicated in DC alterations in genital (pre)neoplastic microenvironment, we identified receptor activator of nuclear factor kappa-B ligand (RANKL), a TNF family member, as a potential candidate. For the first time, we showed that RANKL expression strongly increases during cervical progression. We also confirmed that RANKL is directly secreted by cancer cells and this expression is not related to HPV viral oncoprotein induction. Interestingly, the addition of osteoprotegerin (OPG) in coculture experiments reduces significantly the inhibition of DC maturation, the release of a tolerogenic cytokine profile (IL-12low IL-10high) and the induction of regulatory T (Treg) cells. Our findings suggest that the use of inhibitory molecules directed against RANKL in cervical/vulvar (pre)neoplastic lesions might prevent alterations of DC functionality and represent an attractive strategy to overcome immune tolerance in such cancers. PMID:26155412

The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice.

PubMed

Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang; Liang, Cheng-Guang

2016-01-01

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

GD1a Overcomes Inhibition of Myelination by Fibronectin via Activation of Protein Kinase A: Implications for Multiple Sclerosis.

PubMed

Qin, Jing; Sikkema, Arend H; van der Bij, Kristine; de Jonge, Jenny C; Klappe, Karin; Nies, Vera; Jonker, Johan W; Kok, Jan Willem; Hoekstra, Dick; Baron, Wia

2017-10-11

Remyelination failure by oligodendrocytes contributes to the functional impairment that characterizes the demyelinating disease multiple sclerosis (MS). Since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting remyelination are pivotal in halting disease progression. Our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure by perturbing oligodendrocyte progenitor cell (OPC) maturation. Here, we aim at elucidating whether exogenously added gangliosides (i.e., cell surface lipids with a potential to modulate signaling pathways) could counteract fibronectin-mediated inhibition of OPC maturation. Exclusive exposure of rat oligodendrocytes to GD1a, but not other gangliosides, overcomes aggregated fibronectin-induced inhibition of myelin membrane formation, in vitro , and OPC differentiation in fibronectin aggregate containing cuprizone-induced demyelinated lesions in male mice. GD1a exerts its effect on OPCs by inducing their proliferation and, at a late stage, by modulating OPC maturation. Kinase activity profiling revealed that GD1a activated a protein kinase A (PKA)-dependent signaling pathway and increased phosphorylation of the transcription factor cAMP response element-binding protein. Consistently, the effect of GD1a in restoring myelin membrane formation in the presence of fibronectin aggregates was abolished by the PKA inhibitor H89, whereas the effect of GD1a was mimicked by the PKA activator dibutyryl-cAMP. Together, GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation by activating a PKA-dependent signaling pathway. Given the persistent presence of fibronectin aggregates in MS lesions, ganglioside GD1a might act as a potential novel therapeutic tool to selectively modulate the detrimental signaling environment that precludes remyelination. SIGNIFICANCE STATEMENT As an environmental factor, aggregates of the extracellular matrix protein fibronectin perturb the maturation of oligodendrocyte progenitor cells (OPCs), thereby impeding remyelination, in the demyelinating disease multiple sclerosis (MS). Here we demonstrate that exogenous addition of ganglioside GD1a overcomes the inhibiting effect of aggregated fibronectin on OPC maturation, both in vitro and in vivo , by activating a PKA-dependent signaling pathway. We propose that targeted delivery of GD1a to MS lesions may act as a potential novel molecular tool to boost maturation of resident OPCs to overcome remyelination failure and halt disease progression. Copyright © 2017 the authors 0270-6474/17/379925-14$15.00/0.

Addition of insulin-like growth factor I to the maturation medium of bovine oocytes subjected to heat shock: effects on the production of reactive oxygen species, mitochondrial activity and oocyte competence.

PubMed

Ascari, I J; Alves, N G; Jasmin, J; Lima, R R; Quintão, C C R; Oberlender, G; Moraes, E A; Camargo, L S A

2017-07-01

This study was performed to investigate the effects of insulin-like growth factor-I (IGF-I) addition to in vitro maturation (IVM) medium on apoptosis, mitochondrial membrane potential, ROS production, and developmental competence of bovine oocytes subjected to heat shock. Two temperatures (conventional: 24 h at 38.5°C, or heat shock: 12 h at 41°C followed by 12 h at 38.5°C) and 3 IGF-I concentrations (0, 25, and 100 ng/mL) were tested during IVM. The oocytes were then fertilized in vitro, and the presumptive zygotes were cultured until reaching the blastocyst stage. There was no interaction between temperature and IGF-I concentration for any variable evaluated (P > 0.05). The addition of IGF-I did not alter the proportion of nuclear maturation, TUNEL-positive oocytes and caspase-3 activity, or blastocyst proportion on Days 7 and 8 post-fertilization. Furthermore, the total number of cells and the number of cells in the inner cell mass (ICM) in the blastocyst were not altered (P > 0.05). However, IGF-I increased (P < 0.05) the mitochondrial membrane potential and the production of ROS in oocytes and decreased (P < 0.05) the proportion of apoptotic cells in the ICM in blastocysts. Heat shock increased (P < 0.05) the proportion of TUNEL-positive oocytes and ROS production and reduced (P < 0.05) the mitochondrial membrane potential. Moreover, heat shock increased (P < 0.05) the apoptosis proportion in the ICM cells. In conclusion, supplementing IVM medium with IGF-I may increase the mitochondrial membrane potential and ROS production in oocytes and decrease apoptosis in the ICM in blastocysts. Heat shock for 12 h compromised oocyte developmental competence and increased apoptosis within the ICM cells of the blastocysts. Copyright © 2017 Elsevier Inc. All rights reserved.

BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

PubMed Central

Schemionek, Mirle; Elling, Christian; Steidl, Ulrich; Bäumer, Nicole; Hamilton, Ashley; Spieker, Tilmann; Göthert, Joachim R.; Stehling, Martin; Wagers, Amy; Huettner, Claudia S.; Tenen, Daniel G.; Tickenbrock, Lara; Berdel, Wolfgang E.; Serve, Hubert; Holyoake, Tessa L.; Müller-Tidow, Carsten

2010-01-01

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity. PMID:20053753

Interleukin-6 Directly Impairs the Erythroid Development of Human TF-1 Erythroleukemic Cells

PubMed Central

McCranor, Bryan J.; Kim, Min Jung; Cruz, Nicole M.; Xue, Qian-Li; Berger, Alan E.; Walston, Jeremy D.; Civin, Curt I.; Roy, Cindy N.

2013-01-01

Anemia of inflammation or chronic disease is a highly prevalent form of anemia. The inflammatory cytokine interleukin-6 (IL-6) negatively correlates with hemoglobin concentration in many disease states. The IL-6-hepcidin antimicrobial peptide axis promotes iron-restricted anemia; however the full role of IL-6 in anemia of inflammation is not well-defined. We previously reported that chronic inflammation had a negative impact on maturation of erythroid progenitors in a mouse model. We hypothesized that IL-6 may be responsible for impaired erythropoiesis, independent of iron restriction. To test the hypothesis we utilized the human erythroleukemia TF-1 cell line to model erythroid maturation and exposed them to varying doses of IL-6 over six days. At 10 ng/ml, IL-6 significantly repressed erythropoietin-dependent TF-1 erythroid maturation. While IL-6 did not decrease the expression of genes associated with hemoglobin synthesis, we observed impaired hemoglobin synthesis as demonstrated by decreased benzidine staining. We also observed that IL-6 down regulated expression of the gene SLC4a1 which is expressed late in erythropoiesis. Those findings suggested that IL-6-dependent inhibition of hemoglobin synthesis might occur. We investigated the impact of IL-6 on mitochondria. IL-6 decreased the mitochondrial membrane potential at all treatment doses, and significantly decreased mitochondrial mass at the highest dose. Our studies indicate that IL-6 may impair mitochondrial function in maturing erythroid cells resulting in impaired hemoglobin production and erythroid maturation. Our findings may indicate a novel pathway of action for IL-6 in the anemia of inflammation, and draw attention to the potential for new therapeutic targets that affect late erythroid development. PMID:24119518

Effects of mesenchymal stem cells from human induced pluripotent stem cells on differentiation, maturation, and function of dendritic cells.

PubMed

Gao, Wen-Xiang; Sun, Yue-Qi; Shi, Jianbo; Li, Cheng-Lin; Fang, Shu-Bin; Wang, Dan; Deng, Xue-Quan; Wen, Weiping; Fu, Qing-Ling

2017-03-02

Mesenchymal stem cells (MSCs) have potent immunomodulatory effects on multiple immune cells and have great potential in treating immune disorders. Induced pluripotent stem cells (iPSCs) serve as an unlimited and noninvasive source of MSCs, and iPSC-MSCs have been reported to have more advantages and exhibit immunomodulation on T lymphocytes and natural killer cells. However, the effects of iPSC-MSCs on dendritic cells (DCs) are unclear. The aim of this study is to investigate the effects of iPSC-MSCs on the differentiation, maturation, and function of DCs. Human monocyte-derived DCs were induced and cultured in the presence or absence of iPSC-MSCs. Flow cytometry was used to analyze the phenotype and functions of DCs, and enzyme-linked immunosorbent assay (ELISA) was used to study cytokine production. In this study, we successfully induced MSCs from different clones of human iPSCs. iPSC-MSCs exhibited a higher proliferation rate with less cell senescence than BM-MSCs. iPSC-MSCs inhibited the differentiation of human monocyte-derived DCs by both producing interleukin (IL)-10 and direct cell contact. Furthermore, iPSC-MSCs did not affect immature DCs to become mature DCs, but modulated their functional properties by increasing their phagocytic ability and inhibiting their ability to stimulate proliferation of lymphocytes. More importantly, iPSC-MSCs induced the generation of IL-10-producing regulatory DCs in the process of maturation, which was mostly mediated by a cell-cell contact mechanism. Our results indicate an important role for iPSC-MSCs in the modulation of DC differentiation and function, supporting the clinical application of iPSC-MSCs in DC-mediated immune diseases.

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

PubMed

Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

2015-01-01

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. Copyright© Ferrata Storti Foundation.

Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization.

PubMed

Wang, Feng; Tian, XiuZhi; Zhang, Lu; He, ChangJiu; Ji, PengYun; Li, Yu; Tan, DunXian; Liu, GuoShi

2014-02-01

To analyze the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1, 1.0, or 10.0 μM) as germinal vesicle-stage oocytes. In vitro prospective study. University research laboratory. Animal models for human studies. In vitro culture in the presence of various concentrations of the antioxidant resveratrol. Parameters of hormone levels, oocyte nuclear maturation, cumulus expansion, levels of intracellular glutathione and reactive oxygen species, embryonic cleavage, blastocyst formation, gene expression associated with mature bovine oocytes and cumulus cells, and level of sirtuin 1 gene expression. Resveratrol statistically significantly increased progesterone secretion and decreased estradiol-17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42 mitogen-activated protein kinase (MAPK) cascade in the oocytes. At a concentration of 1.0 μM, resveratrol statistically significantly improved cumulus expansion, polar body formation, the (hatched) blastocyst rate, and the mean number of cells/blastocysts. Meanwhile, resveratrol statistically significantly reduced the level of reactive oxygen species (ROS) and increased the level of glutathione (GSH). For the first time, the expression of the sirtuin-1 gene was identified in granulosa cells, cumulus cells, oocytes, and blastocysts. Further studies revealed that resveratrol promoted sirtuin-1 gene expression. Resveratrol promoted bovine oocyte maturation and subsequent post-in vitro fertilization embryonic development by inducing progesterone secretion and an antioxidant effect, probably in a manner dependent on sirtuin-1. Copyright © 2014 American Society for Reproductive Medicine. All rights reserved.

Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells

PubMed Central

Cao, Nan; Liu, Zumei; Chen, Zhongyan; Wang, Jia; Chen, Taotao; Zhao, Xiaoyang; Ma, Yu; Qin, Lianju; Kang, Jiuhong; Wei, Bin; Wang, Liu; Jin, Ying; Yang, Huang-Tian

2012-01-01

Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells. PMID:22143566

Adjuvant properties of thermal component of hyperthermia enhanced transdermal immunization: effect on dendritic cells.

PubMed

Joshi, Neha; Duhan, Vikas; Lingwal, Neelam; Bhaskar, Sangeeta; Upadhyay, Pramod

2012-01-01

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential.

Inhaled Vitamin D: A Novel Strategy to Enhance Neonatal Lung Maturation.

PubMed

Taylor, Sneha K; Sakurai, Reiko; Sakurai, Tokusho; Rehan, Virender K

2016-12-01

The physiologic vitamin D (VD), 1α,25(OH) 2 D 3 (1,25D) is a local paracrine/autocrine effecter of fetal lung maturation. By stimulating alveolar type II cell and lipofibroblast proliferation and differentiation, parenterally administered 1,25D has been shown to enhance neonatal lung maturation; but due to the potential systemic side effects of the parenteral route, the translational value of these findings might be limited. To minimize the possibility of systemic toxicity, we examined the effects of VD on neonatal lung maturation, when delivered directly to lungs via nebulization. One-day-old rat pups were administered three different doses of 1,25D and its physiologic precursor 25(OH)D (25D), or the diluent, via nebulization daily for 14 days. Pups were sacrificed for lung, kidneys, and blood collection to determine markers of lung maturation, and serum 25D and calcium levels. Compared to controls, nebulized 25D and 1,25D enhanced lung maturation as evidenced by the increased expression of markers of alveolar epithelial (SP-B, leptin receptor), mesenchymal (PPARγ, C/EBPα), and endothelial (VEGF, FLK-1) differentiation, surfactant phospholipid synthesis, and lung morphology without any significant increases in serum 25D and calcium levels. Inhaled VD is a potentially safe and effective novel strategy to enhance neonatal lung maturation.

Effective Targeting of Multiple B-Cell Maturation Antigen-Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells.

PubMed

Friedman, Kevin M; Garrett, Tracy E; Evans, John W; Horton, Holly M; Latimer, Howard J; Seidel, Stacie L; Horvath, Christopher J; Morgan, Richard A

2018-05-01

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

The human sperm epigenome and its potential role in embryonic development.

PubMed

Carrell, Douglas T; Hammoud, Saher Sue

2010-01-01

Along with many of the genome-wide transitions in chromatin composition throughout spermatogenesis, epigenetic modifications on histone tails and DNA are continuously modified to ensure stage specific gene expression in the maturing spermatid. Recent findings have suggested that the repertoire of epigenetic modifications in the mature sperm may have a potential role in the developing embryo and alterations in the epigenetic profile have been associated with infertility. These changes include DNA demethylation and the retention of modified histones at important developmental, signaling and micro-RNA genes, which resemble the epigenetic state of an embryonic stem cell. This review assesses the significance of epigenetic changes during spermatogenesis, and provides insight on recent associations made between altered epigenetic profiles in the mature sperm and its relationship to infertility.

3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing.

PubMed

Takayama, Kazuo; Kawabata, Kenji; Nagamoto, Yasuhito; Kishimoto, Keisuke; Tashiro, Katsuhisa; Sakurai, Fuminori; Tachibana, Masashi; Kanda, Katsuhiro; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

2013-02-01

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Stimulation of GABA-Induced Ca2+ Influx Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Neurons

PubMed Central

Rushton, David J.; Mattis, Virginia B.; Svendsen, Clive N.; Allen, Nicholas D.; Kemp, Paul J.

2013-01-01

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca2+ channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca2+ imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca2+ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca2+ or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca2+ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca2+ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons. PMID:24278369

Suppression of dendritic cells' maturation and functions by daidzein, a phytoestrogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yum, Min Kyu; Jung, Mi Young; Cho, Daeho

2011-12-15

Isoflavones are ubiquitous compounds in foods and in the environment in general. Daidzein and genistein, the best known of isoflavones, are structurally similar to 17{beta}-estradiol and known to exert estrogenic effects. They also evidence a broad variety of biological properties, including antioxidant, anti-carcinogenic, anti-atherogenic and anti-osteoporotic activities. Previously, daidzein was reported to increase the phagocytic activity of peritoneal macrophages and splenocyte proliferation, and to inhibit nitric oxide (NO) production in macrophages. However, its potential impacts on immune response in dendritic cells (DCs), antigen-presenting cells that link innate and adaptive immunity, have yet to be clearly elucidated. In this study, wemore » evaluated the effects of isoflavones on the maturation and activation of DCs. Isoflavones (formononetin, daidzein, equol, biochanin A, genistein) were found to differentially affect the expression of CD86, a costimulatory molecule, on lipopolysaccharide (LPS)-stimulated DCs. In particular, daidzein significantly and dose-dependently inhibited the expression levels of maturation-associated cell surface markers including CD40, costimulatory molecules (CD80, CD86), and major histocompatibility complex class II (I-A{sup b}) molecule on LPS-stimulated DCs. Daidzein also suppressed pro-inflammatory cytokine production such as IL-12p40, IL-6 and TNF-{alpha}, whereas it didn't affect IL-10 and IL-1{beta} expression. Furthermore, daidzein enhanced endocytosis and inhibited the allo-stimulatory ability of LPS-stimulated DCs on T cells, indicating that daidzein treatment can inhibit the functional maturation of DCs. These results demonstrate that daidzein may exhibit immunosuppressive activity by inhibiting the maturation and activation of DCs. -- Highlights: Black-Right-Pointing-Pointer Daidzein inhibited expression of maturation-associated cell surface markers in DCs. Black-Right-Pointing-Pointer Daidzein suppressed expression of pro-inflammatory cytokines in LPS-stimulated DCs. Black-Right-Pointing-Pointer Daidzein enhanced endocytosis and inhibited allo-stimulatory ability of DCs. Black-Right-Pointing-Pointer Daidzein exhibited immunosuppressive activity by inhibiting the activation of DCs.« less

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

PubMed Central

Gaublomme, Jellert; Shekhar, Karthik; Butty, Vincent; Yi, B. Alexander; Kralj, Joel M.; Bloxham, William; Boyer, Laurie A.; Regev, Aviv

2017-01-01

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation. PMID:28333933

Generation, characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells.

PubMed

Zhang, Zhenzhen; Liu, Jianfang; Liu, Yang; Li, Zheng; Gao, Wei-Qiang; He, Zuping

2013-02-01

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end-stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end-stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra-hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra-hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. Copyright © 2012 Wiley Periodicals, Inc.

IL-4/IL-13 Signaling Inhibits the Potential of Early Thymic Progenitors To Commit to the T Cell Lineage.

PubMed

Barik, Subhasis; Miller, Mindy M; Cattin-Roy, Alexis N; Ukah, Tobechukwu K; Chen, Weirong; Zaghouani, Habib

2017-10-15

Early thymic progenitors (ETPs) are endowed with diverse potencies and can give rise to myeloid and lymphoid lineage progenitors. How the thymic environment guides ETP commitment and maturation toward a specific lineage remains obscure. We have previously shown that ETPs expressing the heteroreceptor (HR) comprising IL-4Rα and IL-13Rα1 give rise to myeloid cells but not T cells. In this article, we show that signaling through the HR inhibits ETP maturation to the T cell lineage but enacts commitment toward the myeloid cells. Indeed, HR + ETPs, but not HR - ETPs, exhibit activated STAT6 transcription factor, which parallels with downregulation of Notch1, a critical factor for T cell development. Meanwhile, the myeloid-specific transcription factor C/EBPα, usually under the control of Notch1, is upregulated. Furthermore, in vivo inhibition of STAT6 phosphorylation restores Notch1 expression in HR + ETPs, which regain T lineage potential. In addition, upon stimulation with IL-4 or IL-13, HR - ETPs expressing virally transduced HR also exhibit STAT6 phosphorylation and downregulation of Notch1, leading to inhibition of lymphoid, but not myeloid, lineage potential. These observations indicate that environmental cytokines play a role in conditioning ETP lineage choice, which would impact T cell development. Copyright © 2017 by The American Association of Immunologists, Inc.

Deconstructing Pancreas Developmental Biology

PubMed Central

Benitez, Cecil M.; Goodyer, William R.

2012-01-01

The relentless nature and increasing prevalence of human pancreatic diseases, in particular, diabetes mellitus and adenocarcinoma, has motivated further understanding of pancreas organogenesis. The pancreas is a multifunctional organ whose epithelial cells govern a diversity of physiologically vital endocrine and exocrine functions. The mechanisms governing the birth, differentiation, morphogenesis, growth, maturation, and maintenance of the endocrine and exocrine components in the pancreas have been discovered recently with increasing tempo. This includes recent studies unveiling mechanisms permitting unexpected flexibility in the developmental potential of immature and mature pancreatic cell subsets, including the ability to interconvert fates. In this article, we describe how classical cell biology, genetic analysis, lineage tracing, and embryological investigations are being complemented by powerful modern methods including epigenetic analysis, time-lapse imaging, and flow cytometry-based cell purification to dissect fundamental processes of pancreas development. PMID:22587935

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells

PubMed Central

Scottoni, Federico; Crowley, Claire; Fiadeiro, Rebeca; Maghsoudlou, Panagiotis; Pellegata, Alessandro Filippo; Mazzacuva, Francesca; Gjinovci, Asllan; Lyne, Anne-Marie; Zulini, Justine; Little, Daniel; Mosaku, Olukunbi; Kelly, Deirdre; De Coppi, Paolo; Gissen, Paul

2017-01-01

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications. PMID:29261712

Homeostatic signals do not drive post-thymic T cell maturation.

PubMed

Houston, Evan G; Boursalian, Tamar E; Fink, Pamela J

2012-01-01

Recent thymic emigrants, the youngest T cells in the lymphoid periphery, undergo a 3 week-long period of functional and phenotypic maturation before being incorporated into the pool of mature, naïve T cells. Previous studies indicate that this maturation requires T cell exit from the thymus and access to secondary lymphoid organs, but is MHC-independent. We now show that post-thymic T cell maturation is independent of homeostatic and costimulatory pathways, requiring neither signals delivered by IL-7 nor CD80/86. Furthermore, while CCR7/CCL19,21-regulated homing of recent thymic emigrants to the T cell zones within the secondary lymphoid organs is not required for post-thymic T cell maturation, an intact dendritic cell compartment modulates this process. It is thus clear that, unlike T cell development and homeostasis, post-thymic maturation is focused not on interrogating the T cell receptor or the cell's responsiveness to homeostatic or costimulatory signals, but on some as yet unrecognized property. Copyright © 2012 Elsevier Inc. All rights reserved.

Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

PubMed

Li, Xiaowei; Tzeng, Stephany Y; Liu, Xiaoyan; Tammia, Markus; Cheng, Yu-Hao; Rolfe, Andrew; Sun, Dong; Zhang, Ning; Green, Jordan J; Wen, Xuejun; Mao, Hai-Quan

2016-04-01

Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of Osteogenic Differentiation by Differential Mineralized Matrix Production in Mesenchymal Stromal Cells by Raman Spectroscopy

PubMed Central

Chen, He-Guei; Chiang, Hui-Hua Kenny; Lee, Oscar Kuang-Sheng

2013-01-01

Mesenchymal stromal cells (MSCs) hold great potential in skeletal tissue engineering and regenerative medicine. However, conventional methods that are used in molecular biology to evaluate osteogenic differentiation of MSCs require a relatively large amount of cells. Cell lysis and cell fixation are also required and all these steps are time-consuming. Therefore, it is imperative to develop a facile technique which can provide real-time information with high sensitivity and selectivity to detect the osteogenic maturation of MSCs. In this study, we use Raman spectroscopy as a biosensor to monitor the production of mineralized matrices during osteogenic induction of MSCs. In summary, Raman spectroscopy is an excellent biosensor to detect the extent of maturation level during MSCs-osteoblast differentiation with a non-disruptive, real-time and label free manner. We expect that this study will promote further investigation of stem cell research and clinical applications. PMID:23734254

Enhanced ADCC Activity of Affinity Maturated and Fc-Engineered Mini-Antibodies Directed against the AML Stem Cell Antigen CD96

PubMed Central

Kellner, Christian; Bräutigam, Joachim; Staudinger, Matthias; Schub, Natalie; Peipp, Matthias; Gramatzki, Martin; Humpe, Andreas

2012-01-01

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential. PMID:22879978

Surface proteins and the formation of biofilms by Staphylococcus aureus.

PubMed

Kim, Sung Joon; Chang, James; Rimal, Binayak; Yang, Hao; Schaefer, Jacob

2018-03-01

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [ 15 N]glycine and l-[1- 13 C]threonine, or in separate experiments, by (ii) l-[2- 13 C, 15 N]leucine. We then measured 13 C- 15 N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15 N isotopic enrichments and from the routing of 13 C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro interaction of Stenotrophomonas maltophilia with human monocyte-derived dendritic cells.

PubMed

Roscetto, Emanuela; Vitiello, Laura; Muoio, Rosa; Soriano, Amata A; Iula, Vita D; Vollaro, Antonio; De Gregorio, Eliana; Catania, Maria R

2015-01-01

Stenotrophomonas maltophilia is increasingly identified as an opportunistic pathogen in immunocompromised, cancer and cystic fibrosis (CF) patients. Knowledge on innate immune responses to S. maltophilia and its potential modulation is poor. The present work investigated the ability of 12 clinical S. maltophilia strains (five from CF patients, seven from non-CF patients) and one environmental strain to survive inside human monocyte-derived dendritic cells (DCs). The effects of the bacteria on maturation of and cytokine secretion by DCs were also measured. S. maltophilia strains presented a high degree of heterogeneity in internalization and intracellular replication efficiencies as well as in the ability of S. maltophilia to interfere with normal DCs maturation. By contrast, all S. maltophilia strains were able to activate DCs, as measured by increase in the expression of surface maturation markers and proinflammatory cytokines secretion.

In vitro interaction of Stenotrophomonas maltophilia with human monocyte-derived dendritic cells

PubMed Central

Roscetto, Emanuela; Vitiello, Laura; Muoio, Rosa; Soriano, Amata A.; Iula, Vita D.; Vollaro, Antonio; Gregorio, Eliana De; Catania, Maria R.

2015-01-01

Stenotrophomonas maltophilia is increasingly identified as an opportunistic pathogen in immunocompromised, cancer and cystic fibrosis (CF) patients. Knowledge on innate immune responses to S. maltophilia and its potential modulation is poor. The present work investigated the ability of 12 clinical S. maltophilia strains (five from CF patients, seven from non-CF patients) and one environmental strain to survive inside human monocyte-derived dendritic cells (DCs). The effects of the bacteria on maturation of and cytokine secretion by DCs were also measured. S. maltophilia strains presented a high degree of heterogeneity in internalization and intracellular replication efficiencies as well as in the ability of S. maltophilia to interfere with normal DCs maturation. By contrast, all S. maltophilia strains were able to activate DCs, as measured by increase in the expression of surface maturation markers and proinflammatory cytokines secretion. PMID:26236302

Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons.

PubMed

Telezhkin, Vsevolod; Straccia, Marco; Yarova, Polina; Pardo, Monica; Yung, Sun; Vinh, Ngoc-Nga; Hancock, Jane M; Barriga, Gerardo Garcia-Diaz; Brown, David A; Rosser, Anne E; Brown, Jonathan T; Canals, Josep M; Randall, Andrew D; Allen, Nicholas D; Kemp, Paul J

2018-05-24

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

Post-thymic maturation: young T cells assert their individuality.

PubMed

Fink, Pamela J; Hendricks, Deborah W

2011-07-22

T cell maturation was once thought to occur entirely within the thymus. Now, evidence is mounting that the youngest peripheral T cells in both mice and humans comprise a distinct population from their more mature, yet still naive, counterparts. These cells, termed recent thymic emigrants (RTEs), undergo a process of post-thymic maturation that can be monitored at the levels of cell phenotype and immune function. Understanding this final maturation step in the process of generating useful and safe T cells is of clinical relevance, given that RTEs are over-represented in neonates and in adults recovering from lymphopenia. Post-thymic maturation may function to ensure T cell fitness and self tolerance.

Stem cells for kidney repair: useful tool for acute renal failure?

PubMed

Yokoo, Takashi; Kawamura, Tetsuya; Kobayashi, Eiji

2008-10-01

Several cell types isolated from adult tissues have been reported to differentiate into mature kidney cells that may participate in renal repair after systemic administration. Chen et al. report that local mesenchymal stem cells derived from adult mouse kidneys are another source of cells with similar properties. Although these cells have the potential to differentiate into endothelial-lineage cell types, their therapeutic benefit to the ischemic kidney is mainly via the production of renoprotective factors.

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

PubMed Central

Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

2013-01-01

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

PubMed Central

Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

Mesenchymal Stem Cells and Myeloid Derived Suppressor Cells: Common Traits in Immune Regulation

PubMed Central

Nikolaev, Alexander

2016-01-01

To protect host against immune-mediated damage, immune responses are tightly regulated. The regulation of immune responses is mediated by various populations of mature immune cells, such as T regulatory cells and B regulatory cells, but also by immature cells of different origins. In this review, we discuss regulatory properties and mechanisms whereby two distinct populations of immature cells, mesenchymal stem cells, and myeloid derived suppressor cells mediate immune regulation, focusing on their similarities, discrepancies, and potential clinical applications. PMID:27529074

Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus.

PubMed

Abdi, Khadar; Lai, Chun-Hsiang; Paez-Gonzalez, Patricia; Lay, Mark; Pyun, Joon; Kuo, Chay T

2018-04-25

Specialized, differentiated cells often perform unique tasks that require them to maintain a stable phenotype. Multiciliated ependymal cells (ECs) are unique glial cells lining the brain ventricles, important for cerebral spinal fluid circulation. While functional ECs are needed to prevent hydrocephalus, they have also been reported to generate new neurons: whether ECs represent a stable cellular population remains unclear. Via a chemical screen we found that mature ECs are inherently plastic, with their multiciliated state needing constant maintenance by the Foxj1 transcription factor, which paradoxically is rapidly turned over by the ubiquitin-proteasome system leading to cellular de-differentiation. Mechanistic analyses revealed a novel NF-κB-independent IKK2 activity stabilizing Foxj1 in mature ECs, and we found that known IKK2 inhibitors including viruses and growth factors robustly induced Foxj1 degradation, EC de-differentiation, and hydrocephalus. Although mature ECs upon de-differentiation can divide and regenerate multiciliated ECs, we did not detect evidence supporting EC's neurogenic potential.

Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

PubMed Central

2013-01-01

Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

PubMed

Laskowski, Denise; Båge, Renée; Humblot, Patrice; Andersson, Göran; Sirard, Marc-André; Sjunnesson, Ylva

2017-10-01

Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL - 1 and 0.1 µg mL - 1 ), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation (ATP5D; CYP11A1; NDUFB7; NDUFB10; NDUFS8). Taken together, we hypothesise that the increased proliferation in the insulin-treated groups might impair the developmental potential of the embryos by inducing metabolic stress on the molecular level, which could be detrimental for the survival of the embryo. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.

PubMed

Le Berre-Scoul, Catherine; Chevalier, Julien; Oleynikova, Elena; Cossais, François; Talon, Sophie; Neunlist, Michel; Boudin, Hélène

2017-01-15

Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown. To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron-enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs. We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation. In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron-enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial-derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were mediated in part through purinergic P2Y 1 receptor- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor required for neuronal network maturation. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

A novel enteric neuron–glia coculture system reveals the role of glia in neuronal development

PubMed Central

Le Berre‐Scoul, Catherine; Chevalier, Julien; Oleynikova, Elena; Cossais, François; Talon, Sophie; Neunlist, Michel

2016-01-01

Key points Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown.To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron‐enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs.We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic‐ and glial cell line‐derived neurotrophic factor‐dependent pathways.Using a novel and valuable culture model to study enteric neuron–glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation. Abstract In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron‐enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial‐derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were mediated in part through purinergic P2Y1 receptor‐ and glial cell line‐derived neurotrophic factor‐dependent pathways. Using a novel and valuable culture model to study enteric neuron–glia interactions, our study identified EGCs as a key cellular actor required for neuronal network maturation. PMID:27436013

Homeostatic Signals do not Drive Post-thymic T cell Maturation

PubMed Central

Houston, Evan G.; Boursalian, Tamar E.; Fink, Pamela J.

2012-01-01

Recent thymic emigrants, the youngest T cells in the lymphoid periphery, undergo a 3-week-long period of functional and phenotypic maturation before being incorporated into the pool of mature, naïve T cells. Previous studies indicate that this maturation requires T cell exit from the thymus and access to secondary lymphoid organs, but is MHC-independent. We now show that post-thymic T cell maturation is independent of homeostatic and costimulatory pathways, requiring neither signals delivered by IL-7 nor CD80/86. Furthermore, while CCR7/CCL19,21-regulated homing of recent thymic emigrants to the T cell zones within the secondary lymphoid organs is not required for post-thymic T cell maturation, an intact dendritic cell compartment modulates this process. It is thus clear that, unlike T cell development and homeostasis, post-thymic maturation is focused not on interrogating the T cell receptor or the cell’s responsiveness to homeostatic or costimulatory signals, but on some as yet unrecognized property. PMID:22398309

Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus

PubMed Central

Wolfisberg, Raphael; Kempf, Christoph

2016-01-01

ABSTRACT Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. IMPORTANCE In general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle. PMID:27009963

Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus.

PubMed

Wolfisberg, Raphael; Kempf, Christoph; Ros, Carlos

2016-06-01

Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. In general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

PubMed Central

Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

2011-01-01

Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

Insight into phagocytosis of mature sexual (gametocyte) stages of Plasmodium falciparum using a human monocyte cell line.

PubMed

Bansal, Geetha P; Weinstein, Corey S; Kumar, Nirbhay

2016-05-01

During natural infection malaria parasites are injected into the bloodstream of a human host by the bite of an infected female Anopheles mosquito. Both asexual and mature sexual stages of Plasmodium circulate in the blood. Asexual forms are responsible for clinical malaria while sexual stages are responsible for continued transmission via the mosquitoes. Immune responses generated against various life cycle stages of the parasite have important roles in resistance to malaria and in reducing malaria transmission. Phagocytosis of free merozoites and erythrocytic asexual stages has been well studied, but very little is known about similar phagocytic clearance of mature sexual stages, which are critical for transmission. We evaluated phagocytic uptake of mature sexual (gametocyte) stage parasites by a human monocyte cell line in the absence of immune sera. We found that intact mature stages do not undergo phagocytosis, unless they are either killed or freed from erythrocytes. In view of this observation, we propose that the inability of mature gametocytes to be phagocytized may actually result in malaria transmission advantage. On the other hand, mature gametocytes that are not transmitted to mosquitoes during infection will eventually die and undergo phagocytosis, initiating immune responses that may have transmission blocking potential. A better understanding of early phagocytic clearance and immune responses to gametocytes may identify additional targets for transmission blocking strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

The role of DAMPS in ALA-PDT for skin squamous cell carcinoma (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wang, Xiuli; Wang, Xiaojie; Ji, Jie; Zhang, Haiyan; Shi, Lei

2016-03-01

5-Aminolevulinic acid mediated photodynamic therapy (ALA-PDT) is an established local approach for skin squamous cell carcinoma. It is believed that dangerous signals damage-associated molecular patterns (DAMPs) play an important role in ALA-PDT. In this study, we evaluated in vitro and in vivo expressions of major DAMPs, calreticulin (CRT), heat shock proteins 70 (HSP70), and high mobility group box 1 (HMGB1), induced by ALA-PDT using immunohistochemistry, western blot, and ELISA in a squamous cell carcinoma (SCC) mouse model. The role of DAMPs in the maturation of DCs potentiated by ALA-PDT-treated tumor cells was detected by FACS and ELISA. Our results showed that ALA-PDT enhanced the expression of CRT, HSP70, and HMGB1. These induced DAMPs played an important role in activating DCs by PDT-treated tumor cells, including phenotypic maturation (upregulation of surface expression of MHC-II, CD80, and CD86) and functional maturation (enhanced capability to secrete IFN-γ and IL-12). Furthermore, injecting ALA-PDT-treated tumor cells into naïve mice resulted in complete protection against cancer cells of the same origin. Our findings indicate that ALA-PDT can upregulate DAMPs and enhance tumor immunogenicity, providing a promising strategy for inducing a systemic anticancer immune response.

Intracellular mature IL-37 suppresses tumor metastasis via inhibiting Rac1 activation.

PubMed

Li, Y; Zhao, M; Guo, C; Chu, H; Li, W; Chen, X; Wang, X; Li, Y; Jia, Y; Koussatidjoa, S; Zhu, F; Wang, J; Wang, X; Wang, Q; Zhao, W; Shi, Y; Chen, W; Zhang, L

2018-02-22

IL-37, a newly found anti-inflammatory cytokine of the IL-1 family, has both extracellular and intracellular functions. Accumulating evidences indicate that it is also involved in tumor progression. However, the mechanism and its intracellular target are unclear. In this study, clinical data from 84 patients showed that loss or reduced expression of IL-37 in lung adenocarcinoma tissues was significantly associated with tumor metastasis. We further provided evidence that IL-37 inhibited effectively tumor metastasis in vitro and in vivo. Moreover, we uncovered a novel mechanism by which IL-37 suppressed tumor cell migration via its intracellular mature form (amino acids 46-218). Intracellular mature form of IL-37, but not its extracellular form, markedly inhibited migration of multiple kinds of tumor cells through inhibiting Rac1 activation. Mechanistically, intracellular mature IL-37 directly bound to the CAAX motif in the C-terminal hypervariable region of Rac1, and then inhibited Rac1 membrane translocation and subsequent downstream signaling. Our research identifies intracellular mature IL-37 as a novel endogenous inhibitor of Rac1. Given the crucial roles of Rac1 in tumor angiogenesis and metastasis, intracellular mature IL-37 might serve as a potential strategy for the control of Rac1 activity and tumor progression.

Characterization of dendritic cells in lip and oral cavity squamous cell carcinoma.

PubMed

Costa, Nádia Lago; Gonçalves, Andréia Souza; Martins, Allisson Filipe Lopes; Arantes, Diego Antônio Costa; Silva, Tarcília Aparecida; Batista, Aline Carvalho

2016-07-01

There may be differences in the antitumor immunity induced by dendritic cells (DCs) during the development of squamous cell carcinoma (SCC) located in the lip rather than in the oral cavity. The aim of this study was to evaluate the number of immature and mature DCs in SCC and potentially malignant disorders of the oral cavity and lip. Immunohistochemistry was used to identify the number (cells/mm(2) ) of immature (CD1a(+) ) or mature (CD83(+) ) DCs in samples of oral cavity SCC (OCSCC) (n = 39), lip SCC (LSCC) (n = 23), leukoplakia (LK) (n = 21), actinic cheilitis (AC) (n = 13), and normal mucosa of the oral cavity (OC control, n = 12) and the lip (lip control, n = 11). The number of CD1a(+) cells tended to be higher in the OC control samples compared with the LK (P = 0.04) and OCSCC (P = 0.21). Unlike, this cell population was lower in the lip control than in AC or LSCC (P < 0.05). The number of CD83(+) cells was increased in the LSCC samples compared with the AC and lip control (P = 0.0001) and in OCSCC compared with both the LK (P = 0.001) and OC control (P = 0.0001) samples. LSCC showed an elevated number of CD1a(+) and CD83(+) cells compared with OCSCC (P = 0.03). The population of mature DCs was lower than the population of immature DCs in all of the tested groups (P < 0.05). There were a greater number of both mature and immature DC populations in the LSCC samples than in the OCSCC, which could contribute to establishing a more effective immune antitumor response for this neoplasm. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

PubMed

Meir, Michael; Flemming, Sven; Burkard, Natalie; Bergauer, Lisa; Metzger, Marco; Germer, Christoph-Thomas; Schlegel, Nicolas

2015-10-15

Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling. Copyright © 2015 the American Physiological Society.

Shikonin inhibits maturation of bone marrow-derived dendritic cells and suppresses allergic airway inflammation in a murine model of asthma

PubMed Central

Lee, Chen-Chen; Wang, Chien-Neng; Lai, Yu-Ting; Kang, Jaw-Jou; Liao, Jiunn-Wang; Chiang, Bor-Luen; Chen, Hui-Chen; Cheng, Yu-Wen

2010-01-01

BACKGROUND AND PURPOSE Shikonin exhibits a wide range of anti-inflammatory actions. Here, we assessed its effects on maturation of murine bone marrow-derived dendritic cells (BM-DCs) and on allergic reactions in a murine model of asthma. EXPERIMENTAL APPROACH Cultured murine BM-DCs were used to investigate the effects of shikonin on expression of cell surface markers and their stimulation of T-cell proliferation and cytokine production. The therapeutic potential of shikonin was evaluated in a model of allergic airway disease. KEY RESULTS Shikonin dose-dependently inhibited expression of major histocompatibility complex class II, CD80, CD86, CCR7 and OX40L on BM-DCs, induced by a mixture of ovalbumin (OVA; 100 µg·mL−1) and thymic stromal lymphopoietin (TSLP; 20 ng·mL−1). Shikonin-treated BM-DCs were poor stimulators of CD4+ T lymphocyte and induced lower levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor (TNF)-α release by responding T-cells. After intratracheal instillation of shikonin in OVA-immunized mice, OVA challenge induced lower IL-4, IL-5, IL-13, TNF-α and eotaxin release in bronchial alveolar lavage fluid, lower IL-4 and IL-5 production in lung cells and mediastinal lymph node cells and attenuated OVA-induced lung eosinophilia and airway hyperresponsiveness. CONCLUSION AND IMPLICATIONS Shikonin effectively suppressed OVA + TSLP-induced BM-DC maturation in vitro and inhibited allergic inflammation and airway hyperresponsiveness in a murine model of asthma, showing good potential as a treatment for allergic asthma. Also, our model provides a novel platform for screening drugs for allergic diseases. PMID:20735407

A novel culture method reveals unique neural stem/progenitors in mature porcine iris tissues that differentiate into neuronal and rod photoreceptor-like cells.

PubMed

Royall, Lars N; Lea, Daniel; Matsushita, Tamami; Takeda, Taka-Aki; Taketani, Shigeru; Araki, Masasuke

2017-11-15

Iris neural stem/progenitor cells from mature porcine eyes were investigated using a new protocol for tissue culture, which consists of dispase treatment and Matrigel embedding. We used a number of culture conditions and found an intense differentiation of neuronal cells from both the iris pigmented epithelial (IPE) cells and the stroma tissue cells. Rod photoreceptor-like cells were also observed but mostly in a later stage of culture. Neuronal differentiation does not require any additives such as fetal bovine serum or FGF2, although FGF2 and IGF2 appeared to promote neural differentiation in the IPE cultures. Furthermore, the stroma-derived cells were able to be maintained in vitro indefinitely. The evolutionary similarity between humans and domestic pigs highlight the potential for this methodology in the modeling of human diseases and characterizing human ocular stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

The Formylpeptide Receptor 2 (Fpr2) and Its Endogenous Ligand Cathelin-related Antimicrobial Peptide (CRAMP) Promote Dendritic Cell Maturation*

PubMed Central

Chen, Keqiang; Xiang, Yi; Huang, Jiaqiang; Gong, Wanghua; Yoshimura, Teizo; Jiang, Qun; Tessarollo, Lino; Le, Yingying; Wang, Ji Ming

2014-01-01

Mouse formylpeptide receptor 2 (Fpr2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2, which interacts with pathogen and host-derived chemotactic agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-deficient (Fpr2−/−) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the potential changes in the differentiation and maturation of DCs caused by Fpr2 deficiency. Bone marrow monocytes from Fpr2−/− mouse mice incubated with GM-CSF and IL-4 in vitro showed normal expression of markers of immature DCs. However, upon stimulation with the TLR4 agonist LPS, Fpr2−/− mouse DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2−/− DCs also failed to induce allogeneic T-cell proliferation in vitro, and their recruitment into the T-cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, because neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We additionally observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation. The importance of CRAMP for DC maturation was further demonstrated by the observations that DCs from CRAMP−/− mice expressed lower levels of costimulatory molecules and MHC II and exhibited poor chemotaxis in response to CCL21 after LPS stimulation. Our observations indicate a nonredundant role for Fpr2 and its agonist CRAMP in DC maturation in immune responses. PMID:24808174

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

PubMed Central

Zavašnik-Bergant, Tina; Bergant Marušič, Martina

2016-01-01

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii. PMID:26960148

Increased T Cell Immunosenescence and Accelerated Maturation Phenotypes in Older Kidney Transplant Recipients.

PubMed

Schaenman, J M; Rossetti, M; Sidwell, T; Groysberg, V; Sunga, G; Korin, Y; Liang, E; Zhou, X; Abdallah, B; Lum, E; Bunnapradist, S; Pham, T; Danovitch, G; Reed, E F

2018-06-15

Older kidney transplant recipients experience increased rates of infection and death, and less rejection, compared with younger patients. However, little is known about immune dysfunction in older compared with younger kidney transplant recipients and whether it is associated with infection. We evaluated T cell phenotypes including maturation, immune senescence, and exhaustion in a novel investigation into differences in older compared with younger patients receiving identical immune suppression regimens. We evaluated PBMC from 60 kidney transplant recipients (23 older and 37 matched younger patients) by multiparameter immune phenotyping. Older kidney transplant recipients demonstrated decreased frequency of naïve CD4+ and CD8+ T cells, and increased frequency of terminally differentiated, immune senescent, and NK T cells expressing KLRG1. There was a trend towards increased frequency of T cell immune senescence in patients experiencing infection in the first year after transplantation, which reached statistical significance in a multivariate analysis. This pilot study reveals immune dysfunction in older compared with younger transplant recipients, and suggests a likely mechanism for increased vulnerability to infection. The ability to assess T cell maturation and immune senescence in transplant recipients offers the potential for risk stratification and customization of immune suppression to prevent infection and rejection after transplantation. Copyright © 2018. Published by Elsevier Inc.

Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.

PubMed

De Nichilo, M O; Burns, G F

1993-03-15

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.

Efficacy of Dendritic Cells Matured Early with OK-432 (Picibanil®), Prostaglandin E2, and Interferon-α as a Vaccine for a Hormone Refractory Prostate Cancer Cell Line

PubMed Central

Yoo, Changhee; Do, Hyun-Ah; Jeong, In Gab; Park, Hongzoo; Hwang, Jung-Jin; Hong, Jun Hyuk; Cho, Jin Seon; Choo, Myong-Soo; Ahn, Hanjong

2010-01-01

Dendritic cells (DCs) are potent antigen-presenting cells. OK432 (Picibanil®) was introduced as a potent stimulator of DC maturation in combination with prostaglandin-E2 and interferon-α. We compared the efficacy of a DC-prostate cancer vaccine using early-mature DCs stimulated with OK432, PGE2 and INF-α (OPA) with that of vaccines using other methods. On days 3 or 7 of DC culture, TNF-α (T), TNF-α and LPS (TL) or OPA were employed as maturation stimulators. DU145 cells subjected to heat stress were hybridized with mature DCs using polyethyleneglycol. T cells were sensitized by the hybrids, and their proliferative and cytokine secretion activities and cytotoxicity were measured. The yields of early-mature DCs were higher, compared to yields at the conventional maturation time (P<0.05). In the early maturation setting, the mean fusion ratios, calculated from the fraction of dual-positive cells, were 13.3%, 18.6%, and 39.9%, respectively (P=0.051) in the T only, TL, and OPA-treated groups. The function of cytotoxic T cells, which were sensitized with the hybrids containing DCs matured early with OPA, was superior to that using other methods. The antitumor effects of DC-DU145 hybrids generated with DCs subjected to early maturation with the OPA may be superior to that of the hybrids using conventional maturation methods. PMID:20808670

Efficacy of dendritic cells matured early with OK-432 (Picibanil), prostaglandin E2, and interferon-alpha as a vaccine for a hormone refractory prostate cancer cell line.

PubMed

Yoo, Changhee; Do, Hyun-Ah; Jeong, In Gab; Park, Hongzoo; Hwang, Jung-Jin; Hong, Jun Hyuk; Cho, Jin Seon; Choo, Myong-Soo; Ahn, Hanjong; Kim, Choung-Soo

2010-09-01

Dendritic cells (DCs) are potent antigen-presenting cells. OK432 (Picibanil) was introduced as a potent stimulator of DC maturation in combination with prostaglandin-E(2) and interferon-alpha. We compared the efficacy of a DC-prostate cancer vaccine using early-mature DCs stimulated with OK432, PGE2 and INF-alpha (OPA) with that of vaccines using other methods. On days 3 or 7 of DC culture, TNF-alpha (T), TNF-alpha and LPS (TL) or OPA were employed as maturation stimulators. DU145 cells subjected to heat stress were hybridized with mature DCs using polyethyleneglycol. T cells were sensitized by the hybrids, and their proliferative and cytokine secretion activities and cytotoxicity were measured. The yields of early-mature DCs were higher, compared to yields at the conventional maturation time (P<0.05). In the early maturation setting, the mean fusion ratios, calculated from the fraction of dual-positive cells, were 13.3%, 18.6%, and 39.9%, respectively (P=0.051) in the T only, TL, and OPA-treated groups. The function of cytotoxic T cells, which were sensitized with the hybrids containing DCs matured early with OPA, was superior to that using other methods. The antitumor effects of DC-DU145 hybrids generated with DCs subjected to early maturation with the OPA may be superior to that of the hybrids using conventional maturation methods.

Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system

PubMed Central

Zhu, Huanqi; Scharnhorst, Kelsey S.; Stieg, Adam Z.; Gimzewski, James K.; Minami, Itsunari; Nakatsuji, Norio; Nakano, Haruko; Nakano, Atsushi

2017-01-01

Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals. PMID:28266620

Postthymic maturation influences the CD8 T cell response to antigen.

PubMed

Makaroff, Lydia E; Hendricks, Deborah W; Niec, Rachel E; Fink, Pamela J

2009-03-24

Complete T cell development requires postthymic maturation, and we investigated the influence of this ontological period on the CD8 T cell response to infection by comparing responses of mature CD8 T cells with those of recent thymic emigrants (RTEs). When activated with a noninflammatory stimulus or a bacterial or viral pathogen, CD8 RTEs generated a lower proportion of cytokine-producing effector cells and long-lived memory precursors compared with their mature counterparts. Although peripheral T cell maturation is complete within several weeks after thymic egress, RTE-derived memory cells continued to express inappropriate levels of memory cell markers and display an altered pattern of cytokine production, even 8 weeks after infection. When rechallenged, RTE-derived memory cells generated secondary effector cells that were phenotypically and functionally equivalent to those generated by their mature counterparts. The defects at the effector and memory stages were not associated with differences in the expression of T cell receptor-, costimulation-, or activation-associated cell surface markers yet were associated with lower Ly6C expression levels at the effector stage. This work demonstrates that the stage of postthymic maturation influences cell fate decisions and cytokine profiles of stimulated CD8 T cells, with repercussions that are apparent long after cells have progressed from the RTE compartment.

Interaction of gold nanoparticles and nickel(II) sulfate affects dendritic cell maturation.

PubMed

Deville, Sarah; Baré, Birgit; Piella, Jordi; Tirez, Kristof; Hoet, Peter; Monopoli, Marco P; Dawson, Kenneth A; Puntes, Victor F; Nelissen, Inge

2016-12-01

Despite many investigations have focused on the pristine toxicity of gold nanoparticles (GNPs), little is known about the outcome of co-exposure and interaction of GNPs with heavy metals which can possibly detoxify or potentiate them. Here, the combined exposure of nickel (II) sulfate (NiSO 4 ) and GNPs on the maturation response of dendritic cells (DCs) was explored. Exposure to GNPs or NiSO 4 separately induced cell activation. When cells were exposed to a mixture of both, however, the observed cell activation pattern indicated a competitive rather than an additive effect of both inducers with levels similar to those induced by NiSO 4 alone. Quantification of the GNP uptake by DCs demonstrated a significant decrease in intracellular gold content during co-incubation with NiSO 4 . An extensive physiochemical characterization was performed to determine the interaction between GNPs and NiSO 4 in the complex physiological media using nanoparticle tracking analyses, disc centrifugation, UV-visible spectroscopy, ICP-MS analyses, zeta potential measurements, electron microscopy, and proteomics. Although GNPs and NiSO 4 did not directly interact with each other, the presence of NiSO 4 in the physiological media resulted in changes in GNPs' charge and their associated protein corona (content and composition), which may contribute to a decreased cellular uptake of GNPs and sustaining the nickel-induced DC maturation. The presented results provide new insights in the interaction of heavy metals and NPs in complex physiological media. Moreover, this study highlights the necessity of mixture toxicology, since these combined exposures are highly relevant for human subjection to NPs and risk assessment of nanomaterials.

Prelamin A causes progeria through cell-extrinsic mechanisms and prevents cancer invasion

PubMed Central

de la Rosa, Jorge; Freije, José M. P.; Cabanillas, Rubén; Osorio, Fernando G.; Fraga, Mario F.; Fernández-García, M. Soledad; Rad, Roland; Fanjul, Víctor; Ugalde, Alejandro P.; Liang, Qi; Prosser, Haydn M.; Bradley, Allan; Cadiñanos, Juan; López-Otín, Carlos

2013-01-01

Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A accumulating) and Zmpste24-proficient (mature lamin A containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target. PMID:23917225

Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue.

PubMed

Chen, Yu-Ying; He, Sheng-Teng; Yan, Fu-Hua; Zhou, Peng-Fei; Luo, Kai; Zhang, Yan-Ding; Xiao, Yin; Lin, Min-Kui

2016-12-16

Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue.

Maize development: Cell wall changes in leaves and sheaths

USDA-ARS?s Scientific Manuscript database

Developmental changes occur in maize (Zea mays L.) as it transitions from juvenile stages to the mature plant. Changes also occur as newly formed cells mature into adult cells. Maize leaf blades, including the midribs and sheaths, undergo cell wall changes as cells transition to fully mature cell ty...

Follicular helper T cells in immunity and systemic autoimmunity.

PubMed

Craft, Joseph E

2012-05-01

Follicular helper T (T(FH)) cells are essential for B-cell maturation and immunoglobulin production after immunization with thymus-dependent antigens. Nevertheless, the development and function of T(FH) cells have been less clearly defined than classic CD4(+) effector T-cell subsets, including T-helper-1 (T(H)1), T(H)2 and T(H)17 cells. As such, our understanding of the genesis of T(FH) cells in humans and their role in the development of autoimmunity remains incomplete. However, evidence from animal models of systemic lupus erythematosus (SLE) and patients with systemic autoimmune diseases suggests that these cells are necessary for pathogenic autoantibody production, in a manner analogous to their role in promotion of B-cell maturation during normal immune responses. In this Review, I discuss the findings that have increased our knowledge of T(FH)-cell development and function in normal and aberrant immune responses. Such information might improve our understanding of autoimmune diseases, such as SLE, and highlights the potential of T(FH) cells as therapeutic targets in these diseases.

Diabetic human adipose tissue-derived mesenchymal stem cells fail to differentiate in functional adipocytes.

PubMed

Barbagallo, Ignazio; Li Volti, Giovanni; Galvano, Fabio; Tettamanti, Guido; Pluchinotta, Francesca R; Bergante, Sonia; Vanella, Luca

2017-05-01

Adipose tissue dysfunction represents a hallmark of diabetic patients and is a consequence of the altered homeostasis of this tissue. Mesenchymal stem cells (MSCs) and their differentiation into adipocytes contribute significantly in maintaining the mass and function of adult adipose tissue. The aim of this study was to evaluate the differentiation of MSCs from patients suffering type 2 diabetes (dASC) and how such process results in hyperplasia or rather a stop of adipocyte turnover resulting in hypertrophy of mature adipocytes. Our results showed that gene profile of all adipogenic markers is not expressed in diabetic cells after differentiation indicating that diabetic cells fail to differentiate into adipocytes. Interestingly, delta like 1, peroxisome proliferator-activated receptor alpha, and interleukin 1β were upregulated whereas Sirtuin 1 and insulin receptor substrate 1 gene expression were found downregulated in dASC compared to cells obtained from healthy subjects. Taken together our data indicate that dASC lose their ability to differentiate into mature and functional adipocytes. In conclusion, our in vitro study is the first to suggest that diabetic patients might develop obesity through a hypertrophy of existing mature adipocytes due to failure turnover of adipose tissue. Impact statement In the present manuscript, we evaluated the differentiative potential of mesenchymal stem cells (MSCs) in adipocytes obtained from healthy and diabetic patients. This finding could be of great potential interest for the field of obesity in order to exploit such results to further understand the pathophysiological processes underlying metabolic syndrome. In particular, inflammation in diabetic patients causes a dysfunction in MSCs differentiation and a decrease in adipocytes turnover leading to insulin resistance.

Sex Differences in Maturation of Human Embryonic Stem Cell-Derived β Cells in Mice.

PubMed

Saber, Nelly; Bruin, Jennifer E; O'Dwyer, Shannon; Schuster, Hellen; Rezania, Alireza; Kieffer, Timothy J

2018-04-01

Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate that pancreatic progenitor cells can mature into a mixed population of endocrine cells, including glucose-responsive β cells several months after implantion. However, it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these hESC-derived cells. Here, we investigated the effects of (1) pregnancy on the maturation of human stage 4 (S4) pancreatic progenitor cells and (2) the impact of host sex on both S4 cells and more mature stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-beige mice. Pregnancy led to increased proliferation of endogenous pancreatic β cells, but did not appear to affect proliferation or maturation of S4 cells at midgestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males, whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells before implantation.

Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

PubMed Central

Pezzolla, Daniela; López-Beas, Javier; Lachaud, Christian C.; Domínguez-Rodríguez, Alejandro; Smani, Tarik; Hmadcha, Abdelkrim; Soria, Bernat

2015-01-01

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process. PMID:25774684

Microarray analysis of human milk cells: persistent high expression of osteopontin during the lactation period

PubMed Central

NAGATOMO, T; OHGA, S; TAKADA, H; NOMURA, A; HIKINO, S; IMURA, M; OHSHIMA, K; HARA, T

2004-01-01

To continue the search for immunological roles of breast milk, cDNA microarray analysis on cytokines and growth factors was performed for human milk cells. Among the 240 cytokine-related genes, osteopontin (OPN) gene ranked top of the expression. Real-time PCR revealed that the OPN mRNA levels in colostrum cells were approximately 100 times higher than those in PHA-stimulated peripheral blood mononuclear cells (PBMNCs), and 10 000 times higher than those in PB CD14+ cells. The median levels of OPN mRNA in early milk or mature milk cells were more than three times higher than those in colostrum cells. Western blot analysis of human milk showed appreciable expression of full-length and short form proteins of OPN. The concentrations of full-length OPN in early milk or mature milk whey continued to be higher than those in colostrum whey and plasma as assessed by ELISA. The early milk (3–7 days postpartum) contained the highest concentrations of OPN protein, while the late mature milk cells (1 years postpartum) had the highest expression of OPN mRNA of all the lactating periods. The results of immunohistochemical and immunocytochemical staining indicated that OPN-producing epithelial cells and macrophages are found in actively lactating mammary glands. These results suggest that the persistently and extraordinarily high expression of OPN in human milk cells plays a potential role in the immunological development of breast-fed infants. PMID:15373904

Viscum album neutralizes tumor-induced immunosuppression in a human in vitro cell model

PubMed Central

Steinborn, Carmen; Klemd, Amy Marisa; Sauer, Barbara; Garcia-Käufer, Manuel; Urech, Konrad; Follo, Marie; Ücker, Annekathrin; Kienle, Gunver Sophia; Huber, Roman

2017-01-01

Tumor cells have the capacity to secrete immunosuppressive substances in order to diminish dendritic cell (DC) activity and thereby escape from immune responses. The impact of mistletoe (Viscum album) extracts (VAE), which are frequently used as an additive anti-cancer therapy to stimulate the immune response, is still unknown. Using a human cellular system, the impact of two different VAE (VAEA + VAEI) on the maturation of human dendritic cells and on T cell function has been investigated using flow cytometry, automated fluorescence microscopy and cytokine bead array assays. Furthermore, we examined whether VAEI was able to counteract tumor-induced immunosuppression within this cellular system using a renal cancer cell model. The role of mistletoe lectin (ML) was analyzed using ML-specific antibodies and ML-depleted VAEI. VAEI and VAEA augmented the maturation of dendritic cells. VAEI abrogated tumor-induced immunosuppression of dendritic cells and both processes were partially mediated by ML since ML-depleted VAEI and ML-specific antibodies almost neutralized the rehabilitative effects of VAEI on DC maturation. Using these settings, co-culture experiments with purified CD4+ T cells had no influence on T cell proliferation and activation but did have an impact on IFN-γ secretion. The study provides a potential mode-of-action of VAE as an additive cancer therapy based on immunomodulatory effects. However, the impact on the in vivo situation has to be evaluated in further studies. PMID:28719632

The influence of skeletal maturity on allogenic synovial mesenchymal stem cell-based repair of cartilage in a large animal model.

PubMed

Shimomura, Kazunori; Ando, Wataru; Tateishi, Kosuke; Nansai, Ryosuke; Fujie, Hiromichi; Hart, David A; Kohda, Hideyuki; Kita, Keisuke; Kanamoto, Takashi; Mae, Tatsuo; Nakata, Ken; Shino, Konsei; Yoshikawa, Hideki; Nakamura, Norimasa

2010-11-01

One of the potential factors that may affect the results of mesenchymal stem cell (MSC)-based therapy is the age of donors and recipients. However, there have been no controlled studies to investigate the influence of skeletal maturity on the MSC-based repair of cartilage. The purpose of this study was to compare the repair quality of damaged articular cartilage treated by a scaffold-free three-dimensional tissue-engineered construct (TEC) derived from synovial MSCs between immature and mature pigs. Synovial MSCs were isolated from immature and mature pigs and the proliferation and chondrogenic differentiation capacities were compared. The TEC derived from the synovial MSCs were then implanted into equivalent chondral defects in the medial femoral condyle of both immature and mature pigs, respectively. The implanted defects were morphologically and biomechanically evaluated at 6 months postoperatively. There was no skeletal maturity-dependent difference in proliferation or chondrogenic differentiation capacity of the porcine synovial MSCs. The TEC derived from synovial MSCs promoted the repair of chondral lesion in both immature and mature pigs without the evidence of immune reaction. The repaired tissue by the TEC also exhibited similar viscoelastic properties to normal cartilage regardless of the skeletal maturity. The results of the present study not only suggest the feasibility of allogenic MSC-based cartilage repair over generations but also may validate the use of immature porcine model as clinically relevant to test the feasibility of synovial MSC-based therapies in chondral lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

Down-regulation of RBP-J mediated by microRNA-133a suppresses dendritic cells and functions as a potential tumor suppressor in osteosarcoma.

PubMed

Gao, Xuren; Han, Dong; Fan, Weimin

2016-12-10

In recent years, immunotherapy for the treatment of tumors have been established. Dendritic cells (DCs) are extremely efficient and professional antigen presenting cells (APCs), which are an important target for immune therapeutic interventions in cancer. In present study, we investigated whether RBP-J signaling regulated by miR-133a was involved in the DCs mediated tumor suppressor in osteosarcoma. DCs were isolated from 30 osteosarcoma patients and 30 healthy subjects. Mouse macrophage-like cell line RAW264.7 were cultured and osteosarcoma mouse model with injection of murine osteosarcoma cell line S180 were established. In osteosarcoma patients, miR-133a expression level of DCs was increased, and RBP-J expression in mRNA and protein levels were decreased. MiR-133a inhibitor promoted maturation and activation of DCs in osteosarcoma patients. In osteosarcoma mouse model, miR-133a mimic suppressed the maturation and activation of spleen DCs, while miR-133a inhibitor promoted them. Overexpression of miR-133a decreased therapeutic effect of DCs on osteosarcoma mice. In RAW264.7 cells, miR-133a was observed to target RBP-J and regulate its expression. MiR-133a mimic inhibited the maturation of DCs in cells exposed to LPS, the effect of which was reversed by overexpression of RBP-J. RBP-J mediated by miR-133a probably contributed to the regulation of DCs maturation and activation in osteosarcoma, which functioned as a therapeutic target for the immunotherapy in cancers. Copyright © 2016. Published by Elsevier Inc.

Fibrocyte measurement in peripheral blood correlates with number of cultured mature fibrocytes in vitro and is a potential biomarker for interstitial lung disease in Rheumatoid Arthritis.

PubMed

Just, Søren Andreas; Lindegaard, Hanne; Hejbøl, Eva Kildall; Davidsen, Jesper Rømhild; Bjerring, Niels; Hansen, Søren Werner Karlskov; Schrøder, Henrik Daa; Hansen, Inger Marie Jensen; Barington, Torben; Nielsen, Christian

2017-07-18

Interstitial lung disease (ILD) can be a severe extra-articular disease manifestation in Rheumatoid Arthritis (RA). A potential role of fibrocytes in RA associated ILD (RA-ILD) has not previously been described. We present a modified faster method for measuring circulating fibrocytes, without intracellular staining. The results are compared to the traditional culture method, where the number of monocytes that differentiate into mature fibrocytes in vitro are counted. The results are following compared to disease activity in patients with severe asthma, ILD, RA (without diagnosed ILD) and RA with verified ILD (RA-ILD). CD45 + CD34 + CD11b + (7-AAD - CD3 - CD19 - CD294 - ) cells were isolated by cell sorting and stained for pro-collagen type 1. Thirty-nine patients (10 RA, 9 ILD and 10 with severe asthma, 10 with RA-ILD) and 10 healthy controls (HC) were included. Current medication, disease activity, pulmonary function test and radiographic data were collected. Circulating fibrocytes were quantified by flow cytometry. Peripheral blood mononuclear cells were isolated and cultured for 5 days and the numbers of mature fibrocytes were counted. 90.2% (mean, SD = 1.5%) of the sorted cells were pro-collagen type 1 positive and thereby fulfilled the criteria for being circulating fibrocytes. The ILD and RA-ILD groups had increased levels of circulating fibrocytes compared to HC (p < 0.05). Levels of circulating fibrocytes correlated overall to number of monocytes that subsequently in vitro differentiated to mature fibrocytes (r = 0.81, p < 0.001). RA patients with pathologically reduced diffusion capacity for carbon monoxide adjusted for hemoglobin (DLCO c ) in both the RA and in the combined RA + RA-ILD group, had significantly higher levels of both circulating and number of cultured mature fibrocytes (both p < 0.05). In both groups, the level of circulating fibrocytes and number of mature fibrocytes in culture also correlated to a reduction in DLCO c (r = -0.61 an r = -0.58 both p < 0.05). We presented a fast and valid method for measuring circulating fibrocytes using flow cytometry on lysed peripheral blood. Further, we showed for the first time, that the level of circulating fibrocytes correlated with the number of peripheral blood mononuclear cells, that differentiated into mature fibrocytes in vitro. Reduced DLCO c was correlated with high levels of circulating and mature fibrocytes in RA, which have not been reported previously. In such, this study suggests that fibrocytes may exhibit an important role in the pathogenesis of RA-ILD, which requires further clarification in future studies. ClinicalTrials.gov : NCT02711657 , registered 13/3-2016, retrospectively registered.

To the nucleolar bodies (nucleoli) in cells of the lymphocytic lineage in patients suffering from B - chronic lymphocytic leukemia.

PubMed

Smetana, K; Karban, J; Trneny, M

2010-01-01

The present study was undertaken to provide more information on nucleoli in lymphocytes of B - chronic lymphocytic leukemia. The computer assisted nucleolar and cytoplasmic RNA image densitometry, reflecting the nucleolar and cytoplasmic RNA concentration at the single cell level, demonstrated a remarkable stability during the differentiation and maturation of B- lymphocytes. In contrast, as it was expected, the nucleolar diameter during the lymphocytic development markedly decreased. Thus the nucleolar RNA content of leukemic B-lymphocytes was apparently related to the nucleolar size. In both immature and mature lymphocytes, the cytostatic treatment increased the incidence of micronucleoli, which represent the "inactive" type of nucleoli. However, the decreased values of the nucleolar diameter were statistically significant only in mature lymphocytes of treated patients. On the other hand, despite such observation, it must be mentioned that "large active" and "ring shaped resting" nucleoli were still present in immature and mature lymphocytes after the cytostatic therapy and such cells might represent a potential pool of proliferating cells. As it is generally accepted "large active nucleoli" with multiple fibrillar centers are known to be characteristic for proliferating cells. "Ring shaped resting nucleoli" are present in sleeping cells, which may be stimulated to return to the cell cycle and to proliferate again. In addition, the nucleolar RNA distribution also indicated that Gumprecht ghosts mostly originated from mature lymphocytes. Increased ratio of the nucleolar to cytoplasmic RNA density in Gumprecht ghosts or apoptotic cells and apoptotic bodies of the lymphocytic origin was related to the decreased cytoplasmic RNA concentration. The increased nucleolar size together with the markedly decreased cytoplasmic RNA concentration characteristic for Gumprecht ghosts just reflected the spreading of lymphocytes during smear preparations. In apoptotic cells or bodies of the lymphocytic origin, the "frozen" nucleolar RNA concentration accompanied by a reduced RNA concentration in the cytoplasm exhibited a remarkable similarity to the apoptotic process induced in vitro by the cytostatic treatment. B-chronic lymphocytic leukemia; lymphocytes; nucleolar classes; size; nucleolar RNA image density -concentration.

Measurement of myeloid maturation by flow cytochemistry in HL-60 leukemia: esterase is inducible, myeloperoxidase is not.

PubMed

Ross, D W

1986-05-01

The phenomenon of leukemic cell maturation requires a measurement of myeloid maturation to understand the process and to exploit it as a means of therapy for leukemia. The HL-60 leukemic cell line was used as a model of induced leukemic cell maturation in order to develop a method of quantitating granulocytic and monocytic maturation in response to drug therapy. An automated flow cytochemistry system (Hemalog-D) was employed to measure mean cell volume, myeloperoxidase (MPO), and nonspecific esterase (NSE). For granulocytic maturation induced by vitamin A or DMSO, MPO and cell volume decreased by 50%, maintaining a constant mean cellular MPO concentration throughout maturation from promyelocyte to neutrophil-like forms. For monocytic maturation induced by low-dose ARA-c, the mean NSE increased substantially, while cell volume remained constant. Unlike MPO concentration, NSE was truly inducible and thus a useful quantitative measure of maturation caused by low-dose ARA-c. Flow cytochemistry and cytofluorometry may be developed to allow for quantitative monitoring of therapeutic trials of induced maturation in human leukemias. However, this will require adapting these techniques to the complexity of human leukemias in vivo, and the necessity of handling heterogeneous populations encountered in bone marrow samples.

Cytoskeleton-associated protein 5 and clathrin heavy chain binding regulates spindle assembly in mouse oocytes

PubMed Central

Wang, Dong-Hui; Han, Zhe; Kong, Xiang-Wei; Ma, Yu-Zhen; Yun, Zhi-Zhong; Liang, Cheng-Guang

2017-01-01

Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation. PMID:28177917

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor.

PubMed

Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2016-01-01

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 10(6) hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

PubMed Central

Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2016-01-01

Abstract The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures. PMID:27610270

Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits.

PubMed

Gomis-Rüth, Susana; Stiess, Michael; Wierenga, Corette J; Meyn, Liane; Bradke, Frank

2014-05-01

An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified aromatic-turmerone (AR).

PubMed

Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-10-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone (ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs.

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified Aromatic-Turmerone (AR)

PubMed Central

Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-01-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone(ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs. PMID:23095866

Effects of resource supplements on mature ciliate biofilms: an empirical test using a new type of flow cell.

PubMed

Norf, Helge; Arndt, Hartmut; Weitere, Markus

2009-11-01

Biofilm-dwelling consumer communities play an important role in the matter flux of many aquatic ecosystems. Due to their poor accessibility, little is as yet known about the regulation of natural biofilms. Here, a new type of flow cell is presented which facilitates both experimental manipulation and live observation of natural, pre-grown biofilms. These flow cells were used to study the dynamics of mature ciliate biofilms in response to supplementation of planktonic bacteria. The results suggest that enhanced ciliate productivity could be quickly transferred to micrometazoans (ciliate grazers), making the effects on the standing stock of the ciliates detectable only for a short time. Likewise, no effect on ciliates appeared when micrometazoan consumers were ab initio abundant. This indicates the importance of 'top-down' control of natural ciliate biofilms. The flow cells used here offer great potential for experimentally testing such control mechanisms within naturally cultivated biofilms.

Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

NASA Astrophysics Data System (ADS)

Salick, Max R.

The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

Spectroscopic signature of mouse embryonic stem cell-derived hepatocytes using synchrotron Fourier transform infrared microspectroscopy

NASA Astrophysics Data System (ADS)

Thumanu, Kanjana; Tanthanuch, Waraporn; Ye, Danna; Sangmalee, Anawat; Lorthongpanich, Chanchao; Parnpai, Rangsun; Heraud, Philip

2011-05-01

Stem cell-based therapy for liver regeneration has been proposed to overcome the persistent shortage in the supply of suitable donor organs. A requirement for this to succeed is to find a rapid method to detect functional hepatocytes, differentiated from embryonic stem cells. We propose Fourier transform infrared (FTIR) microspectroscopy as a versatile method to identify the early and last stages of the differentiation process leading to the formation of hepatocytes. Using synchrotron-FTIR microspectroscopy, the means of identifying hepatocytes at the single-cell level is possible and explored. Principal component analysis and subsequent partial least-squares (PLS) discriminant analysis is applied to distinguish endoderm induction from hepatic progenitor cells and matured hepatocyte-like cells. The data are well modeled by PLS with endoderm induction, hepatic progenitor cells, and mature hepatocyte-like cells able to be discriminated with very high sensitivity and specificity. This method provides a practical tool to monitor endoderm induction and has the potential to be applied for quality control of cell differentiation leading to hepatocyte formation.

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

PubMed

Švajger, Urban

2017-04-01

Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Interleukin-7 negatively regulates the development of mature T cells in fetal thymus organ cultures.

PubMed

DeLuca, Dominick; Clark, Dawn R

2002-05-01

We added antibody specific for interleukin-7 (IL-7) to chimeric fetal thymus organ cultures (FTOC) to investigate the involvement of this cytokine at distinct stages of T cell development. We report that the neutralization of IL-7 early in fetal T cell development results in a decrease in the production of mature CD4 or CD8 ('single positive', SP) or CD4/8 negative ('double negative', DN) T cell phenotypes, as defined by their expression of CD3. This loss of T cell development was not complete, but it did include the development of gammadelta T cells. However, if IL-7 was neutralized at later stages of FTOC, the production of CD4/8 positive ('double positive', DP) T cells was increased, and if the addition of the antibody was delayed further, the production of mature SP T cells was increased. This last result could be extended to both alphabeta and gammadelta T cells. These data suggested that IL-7 played a negative regulatory role in the development of progressively mature T cells. Tissue sections of FTOC showed that IL-7 was expressed in the subcapsular region of the tissue where immature T cells reside. However, IL-7 was not detected in the medullary region where mature T cells are located. These data suggest that IL-7 not only supports the development of immature fetal T cells, but it may inhibit the development of mature T cells. The production of mature fetal T cells may, therefore, be delayed until their precursors enter the medullary microenvironment, where IL-7 production is low. In this way, T cells may be prevented from maturing until negative selection or anergy events eliminate or inactivate autoreactive clones.

Maturation of monocyte derived dendritic cells with OK432 boosts IL-12p70 secretion and conveys strong T-cell responses

PubMed Central

2011-01-01

Background Design of tumour specific immunotherapies using the patients' own dendritic cells (DC) is a fast advancing scientific field. The functional qualities of the DC generated in vitro are critical, and today's gold standard for maturation is a cytokine cocktail consisting of IL-1β, IL-6, TNF-α and PGE2 generating cells lacking IL-12p70 production. OK432 is an immunotherapeutic agent derived from killed Streptococcus pyogenes that has been used clinically to treat malignant and benign neoplasms for decades. Methods In this study, we analysed the effects of OK432 on DC maturation, DC migration, cytokine and chemokine secretion as well as T-cell stimulatory capacity, and compared it to the cytokine cocktail alone and combinations of OK432 with the cytokine cocktail. Results OK432 induced a marked up-regulation of CD40 on the cell surface as well as a strong inflammatory response from the DC with significantly more secretion of 19 different cytokines and chemokines compared to the cytokine cocktail. Interestingly, secretion of IL-15 and IL-12p70 was detected at high concentrations after maturation of DC with OK432. However, the OK432 treated DC did not migrate as well as DC treated with cytokine cocktail in a transwell migration assay. During allogeneic T-cell stimulation OK432 treated DC induced proliferation of over 50 percent of CD4 and 30 percent of CD8 T-cells for more than two cell divisions, whereas cytokine cocktail treated DC induced proliferation of 12 and 11 percent of CD4 and CD8 T-cells, respectively. Conclusions The clinically approved compound OK432 has interesting properties that warrants its use in DC immunotherapy and should be considered as a potential immunomodulating agent in cancer immunotherapy. PMID:21208424

Differential requirement for satellite cells during overload-induced muscle hypertrophy in growing versus mature mice.

PubMed

Murach, Kevin A; White, Sarah H; Wen, Yuan; Ho, Angel; Dupont-Versteegden, Esther E; McCarthy, John J; Peterson, Charlotte A

2017-07-10

Pax7+ satellite cells are required for skeletal muscle fiber growth during post-natal development in mice. Satellite cell-mediated myonuclear accretion also appears to persist into early adulthood. Given the important role of satellite cells during muscle development, we hypothesized that the necessity of satellite cells for adaptation to an imposed hypertrophic stimulus depends on maturational age. Pax7 CreER -R26R DTA mice were treated for 5 days with vehicle (satellite cell-replete, SC+) or tamoxifen (satellite cell-depleted, SC-) at 2 months (young) and 4 months (mature) of age. Following a 2-week washout, mice were subjected to sham surgery or 10 day synergist ablation overload of the plantaris (n = 6-9 per group). The surgical approach minimized regeneration, de novo fiber formation, and fiber splitting while promoting muscle fiber growth. Satellite cell density (Pax7+ cells/fiber), embryonic myosin heavy chain expression (eMyHC), and muscle fiber cross sectional area (CSA) were evaluated via immunohistochemistry. Myonuclei (myonuclei/100 mm) were counted on isolated single muscle fibers. Tamoxifen treatment depleted satellite cells by ≥90% and prevented myonuclear accretion with overload in young and mature mice (p < 0.05). Satellite cells did not recover in SC- mice after overload. Average muscle fiber CSA increased ~20% in young SC+ (p = 0.07), mature SC+ (p < 0.05), and mature SC- mice (p < 0.05). In contrast, muscle fiber hypertrophy was prevented in young SC- mice. Muscle fiber number increased only in mature mice after overload (p < 0.05), and eMyHC expression was variable, specifically in mature SC+ mice. Reliance on satellite cells for overload-induced hypertrophy is dependent on maturational age, and global responses to overload differ in young versus mature mice.

Gonadotrophin-inhibitory hormone receptor expression in the chicken pituitary gland: potential influence of sexual maturation and ovarian steroids.

PubMed

Maddineni, S; Ocón-Grove, O M; Krzysik-Walker, S M; Hendricks, G L; Proudman, J A; Ramachandran, R

2008-09-01

Gonadotrophin-inhibitory hormone (GnIH), a hypothalamic RFamide, has been found to inhibit gonadotrophin secretion from the anterior pituitary gland originally in birds and, subsequently, in mammalian species. The gene encoding a transmembrane receptor for GnIH (GnIHR) was recently identified in the brain, pituitary gland and gonads of song bird, chicken and Japanese quail. The objectives of the present study are to characterise the expression of GnIHR mRNA and protein in the chicken pituitary gland, and to determine whether sexual maturation and gonadal steroids influence pituitary GnIHR mRNA abundance. GnIHR mRNA quantity was found to be significantly higher in diencephalon compared to either anterior pituitary gland or ovaries. GnIHR mRNA quantity was significantly higher in the pituitaries of sexually immature chickens relative to sexually mature chickens. Oestradiol or a combination of oestradiol and progesterone treatment caused a significant decrease in pituitary GnIHR mRNA quantity relative to vehicle controls. GnIHR-immunoreactive (ir) cells were identified in the chicken pituitary gland cephalic and caudal lobes. Furthermore, GnIHR-ir cells were found to be colocalised with luteinising hormone (LH)beta mRNA-, or follicle-stimulating hormone (FSH)beta mRNA-containing cells. GnIH treatment significantly decreased LH release from anterior pituitary gland slices collected from sexually immature, but not from sexually mature chickens. Taken together, GnIHR gene expression is possibly down regulated in response to a surge in circulating oestradiol and progesterone levels as the chicken undergoes sexual maturation to allow gonadotrophin secretion. Furthermore, GnIHR protein expressed in FSHbeta or LHbeta mRNA-containing cells is likely to mediate the inhibitory effect of GnIH on LH and FSH secretion.

Bone marrow niche-inspired, multi-phase expansion of megakaryocytic progenitors with high polyploidization potential

PubMed Central

Panuganti, Swapna; Papoutsakis, Eleftherios T.; Miller, William M.

2010-01-01

Background Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization, and platelet release. pH and pO2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks. Methods CD34+ HSPCs were cultured at pH 7.2 and 5% O2 with stem cell factor (SCF), thrombopoietin (Tpo), and all combinations of Interleukin (IL)-3, IL-6, IL-11, and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O2 to generate mature Mks, and treated with nicotinamide to enhance polyploidization. Results Using Tpo+SCF+IL-3+IL-11, we obtained 3.5 CD34+CD41+ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO2. Although fewer progenitors were present, shifting to 20% O2/pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. Nicotinamide more than doubled the percentage of high-ploidy Mks to 40%. Discussion We obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed nicotinamide addition will greatly increase high-ploidy Mk production. PMID:20482285

Bone marrow niche-inspired, multiphase expansion of megakaryocytic progenitors with high polyploidization potential.

PubMed

Panuganti, Swapna; Papoutsakis, Eleftherios T; Miller, William M

2010-10-01

Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks. CD34+ HSPCs were cultured at pH 7.2 and 5% O2 with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O2 to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization. Using Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34+ CD41+ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO2. Although fewer progenitors were present, shifting to 20% O2 /pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%. We obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.

Advances in generation of functional β-cells from induced pluripotent stem cells as a cure for diabetes mellitus.

PubMed

Kalra, Kunal; Chandrabose, Srijaya Thekkeparambil; Ramasamy, Thamil Selvee; Kasim, Noor Hayaty Binti Abu

2018-06-04

Diabetes mellitus is one of the leading cause for death worldwide. Loss and functional failure of pancreatic β-cells, the parenchyma cells in the islets of Langerhans onsets and progresses diabetes mellitus. The increasing incidence of this metabolic disorder necessitates efficient strategies to produce functional β-cells for treating diabetes mellitus. Human induced pluripotent stem cells (hiPSC), holds potential for treating diabetes owning to their self-renewal capacity and ability to differentiate into β-cells. iPSC technology also provides unlimited starting material to generate differentiated cells for regenerative applications. Progress has also been made in establishing in-vitro culture protocols to yield definitive endoderm, pancreatic endoderm progenitor cells and β-cells via different reprogramming strategies and growth factor supplementation. However, these generated β-cells are still immature, lack functional characteristics and exhibit lower capability in reversing the diseases conditions. Current methods employed to generate mature and functional β-cells include; use of small and large molecules to enhance the reprogramming and differentiation efficiency, 3D culture systems to improve the functional properties and heterogeneity of differentiated cells. This review details recent advancements in the generation of mature β-cells by reprogramming stem cells into iPSCs that is further programmed to β-cells. It also provides deeper insight of current reprogramming protocols and their efficacy, focusing on the underlying mechanism of chemical based approach to generate iPSCs. Furthermore, we have highlighted the recent differentiation strategies both in-vitro and in-vivo to date and the future prospects in generation of mature β-cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey.

PubMed

Chen, N; Liow, S-L; Abdullah, R Bin; Embong, W Khadijah Wan; Yip, W-Y; Tan, L-G; Tong, G-Q; Ng, S-C

2007-02-01

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

PubMed

Floren, Michael; Bonani, Walter; Dharmarajan, Anirudh; Motta, Antonella; Migliaresi, Claudio; Tan, Wei

2016-02-01

Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms. This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Chemokine programming dendritic cell antigen response: part II - programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype.

PubMed

Park, Jaehyung; Bryers, James D

2013-05-01

In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1β, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency. © 2012 Blackwell Publishing Ltd.

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

PubMed Central

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development. PMID:23935837

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

PubMed

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

CX3CR1-dependent recruitment of mature NK cells into the central nervous system contributes to control autoimmune neuroinflammation.

PubMed

Hertwig, Laura; Hamann, Isabell; Romero-Suarez, Silvina; Millward, Jason M; Pietrek, Rebekka; Chanvillard, Coralie; Stuis, Hanna; Pollok, Karolin; Ransohoff, Richard M; Cardona, Astrid E; Infante-Duarte, Carmen

2016-08-01

Fractalkine receptor (CX3CR1)-deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild-type and CX3CR1-deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1-deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1-deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b(+) NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4(+) T cells and kill these autoreactive cells more efficiently than the immature CD11b(-) fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differences in Mouse and Human Non-Memory B Cell Pools1

PubMed Central

Benitez, Abigail; Weldon, Abby J.; Tatosyan, Lynnette; Velkuru, Vani; Lee, Steve; Milford, Terry-Ann; Francis, Olivia L.; Hsu, Sheri; Nazeri, Kavoos; Casiano, Carlos M.; Schneider, Rebekah; Gonzalez, Jennifer; Su, Rui-Jun; Baez, Ineavely; Colburn, Keith; Moldovan, Ioana; Payne, Kimberly J.

2014-01-01

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Co-expression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. Here, we validate these markers for identifying analogous subsets in humans and use them to compare the non-memory B cell pools in mice and humans, across tissues, during fetal/neonatal and adult life. Among human CD19+IgM+ B cells, the CD21/CD24 schema identifies distinct populations that correspond to T1 (transitional 1), T2 (transitional 2), FM (follicular mature), and MZ (marginal zone) subsets identified in mice. Markers specific to human B cell development validate the identity of MZ cells and the maturation status of human CD21/CD24 non-memory B cell subsets. A comparison of the non-memory B cell pools in bone marrow (BM), blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the non-memory B cell pool in mouse BM where their frequency is more than twice that in humans. Conversely, in spleen the T1:T2 ratio shows that T2 cells are proportionally ∼8 fold higher in humans than mouse. Despite the relatively small contribution of transitional B cells to the human non-memory pool, the number of naïve FM cells produced per transitional B cell is 3-6 fold higher across tissues than in mouse. These data suggest differing dynamics or mechanisms produce the non-memory B cell compartments in mice and humans. PMID:24719464

N-hexane alters the maturation of oocytes and induces apoptosis in mice.

PubMed

Liu, Jin; Huang, Lei; Sun, Yan; Li, Yu Chen; Zhu, Jian Lin; Wang, Wen Xiang; Zhang, Wen Chang

2013-09-01

This study was aimed to determine the effects of n-hexane on the maturation of mouse oocytes. Cell culture was used to observe the maturation of mouse oocytes and CLSM was employed to determine their apoptosis. Germinal vesicle breakdown (GVBD) and extrusion of the first polar body in mouse oocytes were significantly inhibited by n-hexane. After fertilization, the number of eggs in the mouse was significantly reduced by n-hexane. Mitochondrial membrane potentials (ΔΨm) were altered in mouse oocytes that were leading to apoptosis of the oocytes. N-hexane might have affected the maturation of oocytes, causing alteration of ΔΨm and leading to apoptosis which maybe one of the most important mechanisms. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Workshop summary: New silicon cells

NASA Technical Reports Server (NTRS)

Meulenberg, A.; Iles, P. A.

1993-01-01

The workshop on new silicon cells held during SPRAT12 is summarized. A smaller than average group attended this workshop reflecting the reduction in research dollars available to this portion of the photovoltaics community. Despite the maturity of the silicon technology, a core of the group maintained an excitement about new developments and potential opportunities. The group addressed both the implications and the applications of recent developments. Topics discussed include: light trapping and ultrathin silicon cells; different uses for silicon cells; new silicon cell developments; and radiation tolerant high efficiency cells.

Psychological Stress on Female Mice Diminishes the Developmental Potential of Oocytes: A Study Using the Predatory Stress Model

PubMed Central

Liu, Yu-Xiang; Cheng, Ya-Nan; Miao, Yi-Long; Wei, De-Li; Zhao, Li-Hua; Luo, Ming-Jiu; Tan, Jing-He

2012-01-01

Although the predatory stress experimental protocol is considered more psychological than the restraint protocol, it has rarely been used to study the effect of psychological stress on reproduction. Few studies exist on the direct effect of psychological stress to a female on developmental competence of her oocytes, and the direct effect of predatory maternal stress on oocytes has not been reported. In this study, a predatory stress system was first established for mice with cats as predators. Beginning 24 h after injection of equine chorionic gonadotropin, female mice were subjected to predatory stress for 24 h. Evaluation of mouse responses showed that the predatory stress system that we established increased anxiety-like behaviors and plasma cortisol concentrations significantly and continuously while not affecting food and water intake of the mice. In vitro experiments showed that whereas oocyte maturation and Sr2+ activation or fertilization were unaffected by maternal predatory stress, rate of blastocyst formation and number of cells per blastocyst decreased significantly in stressed mice compared to non-stressed controls. In vivo embryo development indicated that both the number of blastocysts recovered per donor mouse and the average number of young per recipient after embryo transfer of blastocysts with similar cell counts were significantly lower in stressed than in unstressed donor mice. It is concluded that the predatory stress system we established was both effective and durative to induce mouse stress responses. Furthermore, predatory stress applied during the oocyte pre-maturation stage significantly impaired oocyte developmental potential while exerting no measurable impact on nuclear maturation, suggesting that cytoplasmic maturation of mouse oocytes was more vulnerable to maternal stress than nuclear maturation. PMID:23118931

Cytoskeletal stabilization of inhibitory interactions in immunologic synapses of mature human dendritic cells with natural killer cells

PubMed Central

Barreira da Silva, Rosa; Graf, Claudine

2011-01-01

Human mature dendritic cells (DCs) can efficiently stimulate natural killer (NK)–cell responses without being targeted by their cytotoxicity. To understand this important regulatory crosstalk, we characterized the development of the immunologic synapse between mature DCs and resting NK cells. Conjugates between these 2 innate leukocyte populations formed rapidly, persisted for prolonged time periods and matured with DC-derived f-actin polymerization at the synapse. Polarization of IL-12 and IL-12R to the synapse coincided with f-actin polymerization, while other activating and inhibitory molecules were enriched at the interface between DCs and NK cells earlier. Functional assays revealed that inhibition of f-actin polymerization in mature synapses led to an increase of IFN-γ secretion and cytotoxicity by NK cells. This elevated NK-cell reactivity resulted from decreased inhibitory signaling in the absence of MHC class I polarization at the interface, which was observed on inhibition of f-actin polymerization in DCs. Thus, inhibitory signaling is stabilized by f-actin at the synapse between mature DCs and resting NK cells. PMID:21917751

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.

PubMed

Yang, Xiulan; Rodriguez, Marita; Pabon, Lil; Fischer, Karin A; Reinecke, Hans; Regnier, Michael; Sniadecki, Nathan J; Ruohola-Baker, Hannele; Murry, Charles E

2014-07-01

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

PubMed Central

Sun, Xun; Chen, Simu; Han, Jianfeng; Zhang, Zhirong

2012-01-01

Background To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs) more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG) and a series of its mannosylated derivatives. Methods PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs) using flow cytometry. Results PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation. Conclusion These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system. PMID:22745554

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

PubMed

Jochems, Caroline; Fantini, Massimo; Fernando, Romaine I; Kwilas, Anna R; Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Kim, Young-Seung; Brechbiel, Martin W; Gulley, James L; Madan, Ravi A; Heery, Christopher R; Hodge, James W; Newton, Robert; Schlom, Jeffrey; Tsang, Kwong Y

2016-06-21

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation

NASA Astrophysics Data System (ADS)

Granelli-Piperno, Angela; Golebiowska, Angelika; Trumpfheller, Christine; Siegal, Frederick P.; Steinman, Ralph M.

2004-05-01

Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.

Change in the immune function of porcine iliac artery endothelial cells infected with porcine circovirus type 2 and its inhibition on monocyte derived dendritic cells maturation

PubMed Central

Liu, Shiyu; Zou, Zhanming; Zhu, Linlin; Liu, Xinyu; Zhou, Shuanghai

2017-01-01

Porcine circovirus-associated disease is caused by porcine circovirus type 2 (PCV2) infection, which targets iliac artery endothelial cells (PIECs); it leads to severe immunopathologies and is associated with major economic losses in the porcine industry. Here, we report that in vitro PCV2 infection of PIECs causes cell injury, which affects DC function as well as adaptive immunity. Specifically, PCV2 infection downregulated PIEC antigen-presenting molecule expression, upregulated cytokines involved in the immune and inflammatory response causing cell damage and repair, and altered the migratory capacity of PIECs. In addition, PCV2-infected PIECs inhibited DC maturation, enhanced the endocytic ability of DCs, and weakened the stimulatory effect of DCs on T lymphocytes. Together, these findings indicate that profound functional impairment of DCs in the presence of PCV2-infected PIECs may be a potential pathogenic mechanism associated with PCV2-induced porcine disease. PMID:29073194

Engineering retinal progenitor cell and scrollable poly(glycerol-sebacate) composites for expansion and subretinal transplantation

PubMed Central

Redenti, Stephen; Neeley, William L.; Rompani, Santiago; Saigal, Sunita; Yang, Jing; Klassen, Henry; Langer, Robert; Young, Michael J.

2014-01-01

Retinal degenerations cause permanent visual loss and affect millions world-wide. Presently, a novel treatment highlights the potential of using biodegradable polymer scaffolds to induce differentiation and deliver retinal progenitor cells for cell replacement therapy. In this study, we engineered and analyzed a micro-fabricated polymer, poly(glycerol sebacate) (PGS) scaffold, whose useful properties include biocompatibility, elasticity, porosity, and a microtopology conducive to mouse retinal progenitor cell (mRPC) differentiation. In vitro proliferation assays revealed that PGS held up to 86,610 (±9993) mRPCs per square millimeter, which were retained through simulated transplantations. mRPCs adherent to PGS differentiated toward mature phenotypes as evidenced by changes in mRNA, protein levels, and enhanced sensitivity to glutamate. Transplanted composites demonstrated long-term mRPC survival and migrated cells exhibited mature marker expression in host retina. These results suggest that combining mRPCs with PGS scaffolds for subretinal transplantation is a practical strategy for advancing retinal tissue engineering as a restorative therapy. PMID:19361860

Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis.

PubMed

Lee, Seung Tae; Choi, Mun Hwan; Lee, Eun Ju; Gong, Seung Pyo; Jang, Mi; Park, Sang Hyun; Jee, Hyang; Kim, Dae Yong; Han, Jae Yong; Lim, Jeong Mook

2008-11-01

To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. Prospective model study. Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. F1 hybrid B6D2F1 mice. Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. Preimplantation development and stem cell characterization. More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.

Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells

PubMed Central

Kumar, Nathan; Richter, Jenna; Cutts, Josh; Bush, Kevin T; Trujillo, Cleber; Nigam, Sanjay K; Gaasterland, Terry; Brafman, David; Willert, Karl

2015-01-01

The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate. DOI: http://dx.doi.org/10.7554/eLife.08413.001 PMID:26554899

Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

PubMed Central

Osorio, Nancy; Cathala, Laurence; Meisler, Miriam H; Crest, Marcel; Magistretti, Jacopo; Delmas, Patrick

2010-01-01

Cerebellar granule (CG) cells generate high-frequency action potentials that have been proposed to depend on the unique properties of their voltage-gated ion channels. To address the in vivo function of Nav1.6 channels in developing and mature CG cells, we combined the study of the developmental expression of Nav subunits with recording of acute cerebellar slices from young and adult granule-specific Scn8a KO mice. Nav1.2 accumulated rapidly at early-formed axon initial segments (AISs). In contrast, Nav1.6 was absent at early postnatal stages but accumulated at AISs of CG cells from P21 to P40. By P40–P65, both Nav1.6 and Nav1.2 co-localized at CG cell AISs. By comparing Na+ currents in mature CG cells (P66–P74) from wild-type and CG-specific Scn8a KO mice, we found that transient and resurgent Na+ currents were not modified in the absence of Nav1.6 whereas persistent Na+ current was strongly reduced. Action potentials in conditional Scn8a KO CG cells showed no alteration in threshold and overshoot, but had a faster repolarization phase and larger post-spike hyperpolarization. In addition, although Scn8a KO CG cells kept their ability to fire action potentials at very high frequency, they displayed increased interspike-interval variability and firing irregularity in response to sustained depolarization. We conclude that Nav1.6 channels at axon initial segments contribute to persistent Na+ current and ensure a high degree of temporal precision in repetitive firing of CG cells. PMID:20173079

Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission

PubMed Central

Parra-Lobato, Maria C.; Gomez-Jimenez, Maria C.

2011-01-01

After fruit ripening, many fruit-tree species undergo massive natural fruit abscission. Olive (Olea europaea L.) is a stone-fruit with cultivars such as Picual (PIC) and Arbequina (ARB) which differ in mature fruit abscission potential. Ethylene (ET) is associated with abscission, but its role during mature fruit abscission remains largely uncharacterized. The present study investigates the possible roles of ET and polyamine (PA) during mature fruit abscission by modulating genes involved in the ET signalling and biosynthesis pathways in the abscission zone (AZ) of both cultivars. Five ET-related genes (OeACS2, OeACO2, OeCTR1, OeERS1, and OeEIL2) were isolated in the AZ and adjacent cells (AZ–AC), and their expression in various olive organs and during mature fruit abscission, in relation to interactions between ET and PA and the expression induction of these genes, was determined. OeACS2, OeACO2, and OeEIL2 were found to be the only genes that were up-regulated in association with mature fruit abscission. Using the inhibition of ET and PA biosynthesis, it is demonstrated that OeACS2 and OeEIL2 expression are under the negative control of PA while ET induces their expression in AZ–AC. Furthermore, mature fruit abscission depressed nitric oxide (NO) production present mainly in the epidermal cells and xylem of the AZ. Also, NO production was differentially responsive to ET, PA, and different inhibitors. Taken together, the results indicate that PA-dependent ET signalling and biosynthesis pathways participate, at least partially, during mature fruit abscission, and that endogenous NO and 1-aminocyclopropane-1-carboxylic acid maintain an inverse correlation, suggesting an antagonistic action of NO and ET in abscission signalling. PMID:21633085

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

PubMed

Mirzaei, Reza; Saei, Azad; Torkashvand, Fatemeh; Azarian, Bahareh; Jalili, Ahmad; Noorbakhsh, Farshid; Vaziri, Behrouz; Hadjati, Jamshid

2016-08-01

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.

Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.

PubMed

Lessard, Julie; Côté, Julie Anne; Lapointe, Marc; Pelletier, Mélissa; Nadeau, Mélanie; Marceau, Simon; Biertho, Laurent; Tchernof, André

2015-03-07

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Amniotic mesenchymal tissue cells inhibit dendritic cell differentiation of peripheral blood and amnion resident monocytes.

PubMed

Magatti, Marta; De Munari, Silvia; Vertua, Elsa; Nassauto, Claudia; Albertini, Alberto; Wengler, Georg S; Parolini, Ornella

2009-01-01

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G(0) phase and abolishes the production of inflammatory cytokines such as TNF-alpha, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

Age-related differences in articular cartilage wound healing: a potential role for transforming growth factor beta1 in adult cartilage repair.

PubMed

Bos, P K; Verhaar, J A N; van Osch, G J V M

2006-01-01

Objective of this study was to investigate the early wound healing reactions of immature and mature articular cartilage on experimental wound healing in the New Zealand White rabbit. The proliferation potential and glycosaminoglycan production of isolated chondrocytes of these animals was studied in an alginate culture system. A band of tissue with death chondrocytes was observed at wound edges of immature articular cartilage, whereas mature cartilage showed a significant smaller amount of dead chondrocytes. A general increase in TGFbeta1, FGF2 and IGF1 was observed throughout cartilage tissue with the exception of lesion edges. The observed immunonegative area appeared to correlate with the observed cell death in lesion edges. Repair in immature cartilage was indicated by chondrocyte proliferation in clusters and a decrease in defect size. No repair response was observed in mature articular cartilage defects. The alginate culture experiment demonstrated a higher proliferation rate of immature chondrocytes. Addition of recombinant TGFbeta1 increased proliferation rate and GAG production of mature chondrocytes. We were not able to further stimulate immature chondrocytes. These results indicate that TGFbeta1 addition may contribute to induce cartilage repair responses in mature cartilage as observed in immature, developing cartilage.

Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

PubMed

Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

2006-09-15

Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos.

PubMed

Pan, Yangyang; He, Honghong; Cui, Yan; Baloch, Abdul Rasheed; Li, Qin; Fan, Jiangfeng; He, Junfeng; Yu, Sijiu

2015-12-01

This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus-oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

PubMed Central

Pan, Yangyang; He, Honghong; Cui, Yan; Baloch, Abdul Rasheed; Li, Qin; Fan, Jiangfeng; He, Junfeng

2015-01-01

Abstract This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus–oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming. PMID:26655079

Gut-derived commensal bacterial products inhibit liver dendritic cell maturation by stimulating hepatic interleukin-6/signal transducer and activator of transcription 3 activity.

PubMed

Lunz, John G; Specht, Susan M; Murase, Noriko; Isse, Kumiko; Demetris, Anthony J

2007-12-01

Intraorgan dendritic cells (DCs) monitor the environment and help translate triggers of innate immunity into adaptive immune responses. Liver-based DCs are continually exposed, via gut-derived portal venous blood, to potential antigens and bacterial products that can trigger innate immunity. However, somehow the liver avoids a state of perpetual inflammation and protects central immune organs from overstimulation. In this study, we tested the hypothesis that hepatic interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) activity increases the activation/maturation threshold of hepatic DCs toward innate immune signals. The results show that the liver nuclear STAT3 activity is significantly higher than that of other organs and is IL-6-dependent. Hepatic DCs in normal IL-6 wild-type (IL-6(+/+)) mice are phenotypically and functionally less mature than DCs from IL-6-deficient (IL-6(-/-)) or STAT3-inhibited IL-6(+/+) mice, as determined by surface marker expression, proinflammatory cytokine secretion, and allogeneic T-cell stimulation. IL-6(+/+) liver DCs produce IL-6 in response to exposure to lipopolysaccharide (LPS) and cytidine phosphate guanosine oligonucleotides (CpG) but are resistant to maturation compared with IL-6(-/-) liver DCs. Conversely, exogenous IL-6 inhibits LPS-induced IL-6(-/-) liver DC maturation. IL-6/STAT3 signaling influences the liver DC expression of toll-like receptor 9 and IL-1 receptor associated kinase-M. The depletion of gut commensal bacteria in IL-6(+/+) mice with oral antibiotics decreased portal blood endotoxin levels, lowered the expression of IL-6 and phospho-STAT3, and significantly increased liver DC maturation. Gut-derived bacterial products, by stimulating hepatic IL-6/STAT3 signaling, inhibit hepatic DC activation/maturation and thereby elevate the threshold needed for translating triggers of innate immunity into adaptive immune responses. Manipulating gut bacteria may therefore be an effective strategy for altering intrahepatic immune responses.

AHR prevents human IL-1R1hi ILC3 differentiation to natural killer cells

PubMed Central

Hughes, Tiffany; Briercheck, Edward L.; Freud, Aharon G.; Trotta, Rossana; McClory, Susan; Scoville, Steven D.; Keller, Karen; Deng, Youcai; Cole, Jordan; Harrison, Nicholas; Mao, Charlene; Zhang, Jianying; Benson, Don M.; Yu, Jianhua; Caligiuri, Michael A.

2014-01-01

SUMMARY Accumulating evidence indicates that human natural killer (NK) cells develop in secondary lymphoid tissue (SLT) through a so-called “stage 3” developmental intermediate minimally characterized by a CD34-CD117+CD94- immunophenotype that lacks mature NK cell function. This stage 3 population is heterogeneous, potentially composed of functionally distinct innate lymphoid cell (ILC) types that includes interleukin-1 receptor (IL-1R1) positive, IL-22-producing ILC3s. Whether human ILC3s are developmentally related to NK cells is a subject of ongoing investigation. Here we show that antagonism of the aryl hydrocarbon receptor (AHR) or silencing of AHR gene expression promotes differentiation of tonsillar IL-22-producing IL-1R1hi human ILC3s to CD56brightCD94+ IFN-gamma-producing cytolytic mature NK cells expressing eomesodermin (EOMES) and T-Box Protein 21 (TBX21 or TBET). Hence, AHR is a transcription factor that prevents human IL-1R1hi ILC3s from differentiating into NK cells. PMID:24953655

Examination of relaxin and its receptors expression in pig gametes and embryos

PubMed Central

2011-01-01

Background Relaxin is a small peptide also known as pregnancy hormone in many mammals. It is synthesized by both male and female tissues, and its secretions are found in various body fluids such as plasma serum, ovarian follicular fluid, utero-oviduct secretions, and seminal plasma of many mammals, including pigs. However, the presence and effects of relaxin in porcine gametes and embryos are still not well-known. The purpose of this study was to assess the presence of relaxin and its receptors RXFP1 and RXFP2 in pig gametes and embryos. Methods Immature cumulus-oocyte complexes (COCs) were aspirated from sows' ovaries collected at the abattoir. After in vitro-maturation, COCs were in vitro-fertilized and cultured. For studies, immature and mature COCs were separately collected, and oocytes were freed from their surrounding cumulus cells. Denuded oocytes, cumulus cells, mature boar spermatozoa, zygotes, and embryos (cleaved and blastocysts) were harvested for temporal and spatial gene expression studies. Sections of ovary, granulosa and neonatal porcine uterine cells were also collected to use as controls. Results Using both semi-quantitative and quantitative PCRs, relaxin transcripts were not detected in all tested samples, while RXFP1 and RXFP2 mRNA were present. Both receptor gene products were found at higher levels in oocytes compared to cumulus cells, irrespective of the maturation time. Cleaved-embryos contained higher levels of RXFP2 mRNA, whereas, blastocysts were characterized by a higher RXFP1 mRNA content. Using western-immunoblotting or in situ immunofluorescence, relaxin and its receptor proteins were detected in all samples. Their fluorescence intensities were consistently more important in mature oocytes than immature ones. The RXFP1 and RXFP2 signal intensities were mostly located in the plasma membrane region, while the relaxin ones appeared homogeneously distributed within the oocytes and embryonic cells. Furthermore, spermatozoa displayed stronger RXFP2 signal than RXFP1 after western-immunoblotting. Conclusion All together, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond. PMID:21251292

c-Met and its ligand hepatocyte growth factor/scatter factor regulate mature B cell survival in a pathway induced by CD74.

PubMed

Gordin, Maya; Tesio, Melania; Cohen, Sivan; Gore, Yael; Lantner, Frida; Leng, Lin; Bucala, Richard; Shachar, Idit

2010-08-15

The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74-HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response.

Microsystems Enabled Photovoltaics

ScienceCinema

Gupta, Vipin; Nielson, Greg; Okandan, Murat, Granata, Jennifer; Nelson, Jeff; Haney, Mike; Cruz-Campa, Jose Luiz

2018-06-07

Sandia's microsystems enabled photovoltaic advances combine mature technology and tools currently used in microsystem production with groundbreaking advances in photovoltaics cell design, decreasing production and system costs while improving energy conversion efficiency. The technology has potential applications in buildings, houses, clothing, portable electronics, vehicles, and other contoured structures.

Induction of Oligodendrocyte Differentiation and In Vitro Myelination by Inhibition of Rho-Associated Kinase

PubMed Central

Taylor, Christopher; Pereira, Albertina; Seng, Michelle; Tham, Chui-Se; Izrael, Michal; Webb, Michael

2014-01-01

In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies. PMID:25289646

Cellular Components, Including Stem-Like Cells, of Preterm Mother's Mature Milk as Compared with Those in Her Colostrum: A Pilot Study.

PubMed

Kaingade, Pankaj; Somasundaram, Indumathi; Sharma, Akshita; Patel, Darshan; Marappagounder, Dhanasekaran

2017-09-01

Whether the preterm mothers' mature milk retains the same cellular components as those in colostrum including stem-like cell, cell adhesion molecules, and immune cells. A total of five preterm mothers were recruited for the study having an average age of 30.2 years and gestational age of 29.8 weeks from the Pristine Women's Hospital, Kolhapur. Colostrum milk was collected within 2-5 days and matured milk was collected 20-30 days after delivery from the same mothers. Integral cellular components of 22 markers including stem cells, immune cells, and cell adhesion molecules were measured using flowcytometry. Preterm mature milk was found to possess higher expressions of hematopoietic stem cells, mesenchymal stem-like cells, immune cells, few cell adhesion molecules, and side population cells than colostrum. The increased level of these different cell components in mature milk may be important in the long-term preterm baby's health growth. Further similar research in a larger population of various gestational ages and lactation stages of preterm mothers is warranted to support these pilot findings.

Investigation of biochemical property changes in activation-induced CD 8 + T cell apoptosis using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Lee, Young Ju; Ahn, Hyung Joon; Lee, Gi-Ja; Jung, Gyeong Bok; Lee, Gihyun; Kim, Dohyun; Shin, Jae-Ho; Jin, Kyung-Hyun; Park, Hun-Kuk

2015-07-01

The study was to investigate the changes in biochemical properties of activated mature CD8+ T cells related to apoptosis at a molecular level. We confirmed the activation and apoptosis of CD8+ T cells by fluorescence-activated cell sorting and atomic force microscopy and then performed Raman spectral measurements on activated mature CD8+ T cells and cellular deoxyribose nucleic acid (DNA). In the activated mature CD8+ T cells, there were increases in protein spectra at 1002 and 1234 cm-1. In particular, to assess the apoptosis-related DNA spectral signatures, we investigated the spectra of the cellular DNA isolated from resting and activated mature CD8+ T cells. Raman spectra at 765 to 786 cm-1 and 1053 to 1087 cm-1 were decreased in activated mature DNA. In addition, we analyzed Raman spectrum using the multivariate statistical method including principal component analysis. Raman spectra of activated mature DNA are especially well-discriminated from those of resting DNA. Our findings regarding the biochemical and structural changes associated with apoptosis in activated mature T cells and cellular DNA according to Raman spectroscopy provide important insights into allospecific immune responses generated after organ transplantation, and may be useful for therapeutic manipulation of the immune response.

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms.

PubMed

Heair, Hannah M; Kemper, Austin G; Roy, Bhaskar; Lopes, Helena B; Rashid, Harunur; Clarke, John C; Afreen, Lubana K; Ferraz, Emanuela P; Kim, Eddy; Javed, Amjad; Beloti, Marcio M; MacDougall, Mary; Hassan, Mohammad Q

2015-09-01

Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

PubMed Central

Puchner, Elias M.; Walter, Jessica M.; Kasper, Robert; Huang, Bo; Lim, Wendell A.

2013-01-01

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes. PMID:24043832

Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster.

PubMed

Wüst, Stas; Dröse, Stefan; Heidler, Juliana; Wittig, Ilka; Klockner, Ina; Franko, Andras; Bonke, Erik; Günther, Stefan; Gärtner, Ulrich; Boettger, Thomas; Braun, Thomas

2018-05-01

Muscle stem cells undergo a dramatic metabolic switch to oxidative phosphorylation during differentiation, which is achieved by massively increased mitochondrial activity. Since expression of the muscle-specific miR-1/133a gene cluster correlates with increased mitochondrial activity during muscle stem cell (MuSC) differentiation, we examined the potential role of miR-1/133a in metabolic maturation of skeletal muscles in mice. We found that miR-1/133a downregulate Mef2A in differentiated myocytes, thereby suppressing the Dlk1-Dio3 gene cluster, which encodes multiple microRNAs inhibiting expression of mitochondrial genes. Loss of miR-1/133a in skeletal muscles or increased Mef2A expression causes continuous high-level expression of the Dlk1-Dio3 gene cluster, compromising mitochondrial function. Failure to terminate the stem cell-like metabolic program characterized by high-level Dlk1-Dio3 gene cluster expression initiates profound changes in muscle physiology, essentially abrogating endurance running. Our results suggest a major role of miR-1/133a in metabolic maturation of skeletal muscles but exclude major functions in muscle development and MuSC maintenance. Copyright © 2018 Elsevier Inc. All rights reserved.

Regulation of Breast Cancer Stem Cell by Tissue Rigidity

DTIC Science & Technology

2014-06-01

pose the similar question as Paszek et al but in a more biomimetic niche: “Does the mature mammary acinar structure desensitize mammary epithelial...2728032. 6. Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol Life Sci. 2006;63(19-20):2304-16. 7. Levental...requirement of EMT and/or MET during the individual steps of tumor metastasis and discuss the potential of targeting this program when treating

On the occurrence of nuclei in mature sieve elements.

PubMed

Event, R F; Davis, J D; Tucker, C M; Alfieri, F J

1970-12-01

The secondary phloem of 3 species of the Taxodiaceae and 13 species of woody dicotyledons was examined for the occurrence of nuclei in mature sieve elements. Nuclei were found in all mature sieve cells of Metasequoia glyptostroboides, Sequoia sempervirens and Taxodium distichum, and in some mature sieve-tube members in 12 of the 13 species of woody dicotyledons. Except for nuclei of sieve cells undergoing cessation of function, the nuclei in mature sieve cells of M. glyptostroboides, S. sempervirens and T. distichum were normal in appearance. The occurrence and morphology of nuclei in mature sieve-tube members of the woody dicotyledons were quite variable. Only 3 species, Robinia pseudoacacia, Ulmus americana and Vitis riparia, contained some mature sieve elements with apparently normal nuclei.

Development of model for analysing respective collections of intended hematopoietic stem cells and harvests of unintended mature cells in apheresis for autologous hematopoietic stem cell collection.

PubMed

Hequet, O; Le, Q H; Rodriguez, J; Dubost, P; Revesz, D; Clerc, A; Rigal, D; Salles, G; Coiffier, B

2014-04-01

Hematopoietic stem cells (HSCs) required to perform peripheral hematopoietic autologous stem cell transplantation (APBSCT) can be collected by processing several blood volumes (BVs) in leukapheresis sessions. However, this may cause granulocyte harvest in graft and decrease in patient's platelet blood level. Both consequences may induce disturbances in patient. One apheresis team's current purpose is to improve HSC collection by increasing HSC collection and prevent increase in granulocyte and platelet harvests. Before improving HSC collection it seemed important to know more about the way to harvest these types of cells. The purpose of our study was to develop a simple model for analysing respective collections of intended CD34+ cells among HSC (designated here as HSC) and harvests of unintended platelets or granulocytes among mature cells (designated here as mature cells) considering the number of BVs processed and factors likely to influence cell collection or harvest. For this, we processed 1, 2 and 3 BVs in 59 leukapheresis sessions and analysed corresponding collections and harvests with a referent device (COBE Spectra). First we analysed the amounts of HSC collected and mature cells harvested and second the evolution of the respective shares of HSC and mature cells collected or harvested throughout the BV processes. HSC collections and mature cell harvests increased globally (p<0.0001) and their respective shares remained stable throughout the BV processes (p non-significant). We analysed the role of intrinsic (patient's features) and extrinsic (features before starting leukapheresis sessions) factors in collections and harvests, which showed that only pre-leukapheresis blood levels (CD34+cells and platelets) influenced both cell collections and harvests (CD34+cells and platelets) (p<0.001) and shares of HSC collections and mature unintended cells harvests (p<0.001) throughout the BV processes. Altogether, our results suggested that the main factors likely to influence intended HSC collections or unintended mature cell harvests were pre-leukapheresis blood cell levels. Our model was meant to assist apheresis teams in analysing shares of HSC collected and mature cells harvested with new devices or with new types of HSC mobilization. Copyright © 2014 Elsevier Ltd. All rights reserved.

Erythropoiesis from Human Embryonic Stem Cells Through Erythropoietin-Independent AKT Signaling

PubMed Central

Kim, William S.; Zhu, Yuhua; Deng, Qiming; Chin, Chee Jia; He, Chong Bin; Grieco, Amanda J.; Dravid, Gautam G.; Parekh, Chintan; Hollis, Roger P.; Lane, Timothy F.; Bouhassira, Eric E.; Kohn, Donald B.; Crooks, Gay M.

2014-01-01

Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homo-dimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of the present study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESC) and to identify the signaling pathways activated by this strategy. We present here evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates the GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC. PMID:24677652

Oligodendrocyte progenitor programming and reprogramming: Toward myelin regeneration.

PubMed

Lopez Juarez, Alejandro; He, Danyang; Richard Lu, Q

2016-05-01

Demyelinating diseases such as multiple sclerosis (MS) are among the most disabling and cost-intensive neurological disorders. The loss of myelin in the central nervous system, produced by oligodendrocytes (OLs), impairs saltatory nerve conduction, leading to motor and cognitive deficits. Immunosuppression therapy has a limited efficacy in MS patients, arguing for a paradigm shift to strategies that target OL lineage cells to achieve myelin repair. The inhibitory microenvironment in MS lesions abrogates the expansion and differentiation of resident OL precursor cells (OPCs) into mature myelin-forming OLs. Recent studies indicate that OPCs display a highly plastic ability to differentiate into alternative cell lineages under certain circumstances. Thus, understanding the mechanisms that maintain and control OPC fate and differentiation into mature OLs in a hostile, non-permissive lesion environment may open new opportunities for regenerative therapies. In this review, we will focus on 1) the plasticity of OPCs in terms of their developmental origins, distribution, and differentiation potentials in the normal and injured brain; 2) recent discoveries of extrinsic and intrinsic factors and small molecule compounds that control OPC specification and differentiation; and 3) therapeutic potential for motivation of neural progenitor cells and reprogramming of differentiated cells into OPCs and their likely impacts on remyelination. OL-based therapies through activating regenerative potentials of OPCs or cell replacement offer exciting opportunities for innovative strategies to promote remyelination and neuroprotection in devastating demyelinating diseases like MS. This article is part of a Special Issue entitled SI:NG2-glia(Invited only). Copyright © 2015 Elsevier B.V. All rights reserved.

Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

PubMed

Zhang, W; Kong, C W; Tong, M H; Chooi, W H; Huang, N; Li, R A; Chan, B P

2017-02-01

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study. Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as being a promising source of cells for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. In the current study, we have fabricated cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials and demonstrated that supplementation of mesenchymal niche cells as well as provision of mechanical loading particularly stretching have significantly promoted the maturation of the cardiomyocytes and hence improved the mechanical functional characteristics of the tissue strips. Specifically, with 3% niche cells including both fibroblasts and mesenchymal stem cells, a more mature hESC-CMs derived cardiac strip was resulted, in terms of compaction and spreading of cells, and upregulation of molecular signature in both gene and protein expression of maturation. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of molecular signature markers and functional parameters including twitch force, elastic modulus and sarcomere length, when comparing with static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture, resulting in more mature cardiac strips. Our results contribute to bioengineering of functional heart tissue strips for drug screening and disease modeling. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Pharmacological approaches to intervention in hypomyelinating and demyelinating white matter pathology

PubMed Central

Chew, Li-Jin; DeBoy, Cynthia A

2015-01-01

White matter disease afflicts both developing and mature central nervous systems. Both cell intrinsic and extrinsic dysregulation result in profound changes in cell survival, axonal metabolism and functional performance. Experimental models of developmental white matter (WM) injury and demyelination have not only delineated mechanisms of signaling and inflammation, but have also paved the way for the discovery of pharmacological approaches to intervention. These reagents have been shown to enhance protection of the mature oligodendrocyte cell, accelerate progenitor cell recruitment and/or differentiation, or attenuate pathological stimuli arising from the inflammatory response to injury. Here we highlight reports of studies in the CNS in which compounds, namely peptides, hormones, and small molecule agonists/antagonists, have been used in experimental animal models of demyelination and neonatal brain injury that affect aspects of excitotoxicity, oligodendrocyte development and survival, and progenitor cell function, and which have been demonstrated to attenuate damage and improve WM protection in experimental models of injury. The molecular targets of these agents include growth factor and neurotransmitter receptors, morphogens and their signaling components, nuclear receptors, as well as the processes of iron transport and actin binding. By surveying the current evidence in non-immune targets of both the immature and mature WM, we aim to better understand pharmacological approaches modulating endogenous oligodendroglia that show potential for success in the contexts of developmental and adult WM pathology. PMID:26116759

Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells

PubMed Central

Mobergslien, Anne; Vasovic, Vlada; Mathiesen, Geir; Fredriksen, Lasse; Westby, Phuong; Eijsink, Vincent GH; Peng, Qian; Sioud, Mouldy

2015-01-01

Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies. PMID:26185907

Efficient Generation of Human Embryonic Stem Cell-Derived Cardiac Progenitors Based on Tissue-Specific Enhanced Green Fluorescence Protein Expression

PubMed Central

Szebényi, Kornélia; Péntek, Adrienn; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I.; Sarkadi, Balázs

2015-01-01

Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFPhigh rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFPhigh rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFPhigh rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications. PMID:24734786

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

PubMed

El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

MHC drives TCR repertoire shaping, but not maturation, in recent thymic emigrants.

PubMed

Houston, Evan G; Fink, Pamela J

2009-12-01

After developing in the thymus, recent thymic emigrants (RTEs) enter the lymphoid periphery and undergo a maturation process as they transition into the mature naive (MN) T cell compartment. This maturation presumably shapes RTEs into a pool of T cells best fit to function robustly in the periphery without causing autoimmunity; however, the mechanism and consequences of this maturation process remain unknown. Using a transgenic mouse system that specifically labels RTEs, we tested the influence of MHC molecules, key drivers of intrathymic T cell selection and naive peripheral T cell homeostasis, in shaping the RTE pool in the lymphoid periphery. We found that the TCRs expressed by RTEs are skewed to longer CDR3 regions compared with those of MN T cells, suggesting that MHC does streamline the TCR repertoire of T cells as they transition from the RTE to the MN T cell stage. This conclusion is borne out in studies in which the representation of individual TCRs was followed as a function of time since thymic egress. Surprisingly, we found that MHC is dispensable for the phenotypic and functional maturation of RTEs.

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

PubMed Central

Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

2015-01-01

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation. PMID:25934799

Accelerating bioelectric functional development of neural stem cells by graphene coupling: Implications for neural interfacing with conductive materials.

PubMed

Guo, Rongrong; Zhang, Shasha; Xiao, Miao; Qian, Fuping; He, Zuhong; Li, Dan; Zhang, Xiaoli; Li, Huawei; Yang, Xiaowei; Wang, Ming; Chai, Renjie; Tang, Mingliang

2016-11-01

In order to govern cell-specific behaviors in tissue engineering for neural repair and regeneration, a better understanding of material-cell interactions, especially the bioelectric functions, is extremely important. Graphene has been reported to be a potential candidate for use as a scaffold and neural interfacing material. However, the bioelectric evolvement of cell membranes on these conductive graphene substrates remains largely uninvestigated. In this study, we used a neural stem cell (NSC) model to explore the possible changes in membrane bioelectric properties - including resting membrane potentials and action potentials - and cell behaviors on graphene films under both proliferation and differentiation conditions. We used a combination of single-cell electrophysiological recordings and traditional cell biology techniques. Graphene did not affect the basic membrane electrical parameters (capacitance and input resistance), but resting membrane potentials of cells on graphene substrates were more strongly negative under both proliferation and differentiation conditions. Also, NSCs and their progeny on graphene substrates exhibited increased firing of action potentials during development compared to controls. However, graphene only slightly affected the electric characterizations of mature NSC progeny. The modulation of passive and active bioelectric properties on the graphene substrate was accompanied by enhanced NSC differentiation. Furthermore, spine density, synapse proteins expressions and synaptic activity were all increased in graphene group. Modeling of the electric field on conductive graphene substrates suggests that the electric field produced by the electronegative cell membrane is much higher on graphene substrates than that on control, and this might explain the observed changes of bioelectric development by graphene coupling. Our results indicate that graphene is able to accelerate NSC maturation during development, especially with regard to bioelectric evolvement. Our findings provide a fundamental understanding of the role of conductive materials in tuning the membrane bioelectric properties in a graphene model and pave the way for future studies on the development of methods and materials for manipulating membrane properties in a controllable way for NSC-based therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greene, Carol Ann, E-mail: carol.greene@auckland.ac.nz; Chang, Chuan-Yuan; Fraser, Cameron J.

Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. Themore » switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment. - Highlights: • Adult corneal stromal cells can differentiated into neuron-like cells. • Neuronal specification of the adult stromal cell population is stochastic. • Neuronal specification in an adult cell population can be brought about by growth factors.« less

PLC-beta2 monitors the drug-induced release of differentiation blockade in tumoral myeloid precursors.

PubMed

Brugnoli, Federica; Bovolenta, Matteo; Benedusi, Mascia; Miscia, Sebastianó; Capitani, Silvano; Bertagnolo, Valeria

2006-05-01

The differentiation therapy in treatment of acute promyelocytic leukemia (APL), based on the administration of all-trans retinoic acid (ATRA), is currently flanked with the use of As2O3, a safe and effective agent for patients showing a resistance to ATRA treatment. A synergy between ATRA and As3O3 was also reported in inducing granulocytic differentiation of APL-derived cells. We have demonstrated that phospholipase C-beta2 (PLC-beta2), highly expressed in neutrophils and nearly absent in tumoral promyelocytes, largely increases during ATRA treatment of APL-derived cells and strongly correlates with the responsiveness of APL patients to ATRA-based differentiating therapies. Here we report that, in APL-derived cells, low doses of As3O3 induce a slight increase of PLC-beta2 together with a moderate maturation, and cooperate with ATRA to provoke a significant increase of PLC-beta2 expression. Remarkably, the amounts of PLC-beta2 draw a parallel with the differentiation levels reached by both ATRA-responsive and -resistant cells treated with ATRA/As2O3 combinations. PLC-beta2 is not necessary for the progression of tumoral promyelocytes along the granulocytic lineage and is unable to overcome the differentiation block or to potentiate the agonist-induced maturation. On the other hand, since its expression closely correlates with the differentiation level reached by APL-derived cells induced to maturate by drugs presently employed in APL therapies, PLC-beta2 represents indeed a specific marker to test the ability of differentiation agents to induce the release of the maturation blockade of tumoral myeloid precursors.

Biophysical Properties and Motility of Human Mature Dendritic Cells Deteriorated by Vascular Endothelial Growth Factor through Cytoskeleton Remodeling

PubMed Central

Hu, Zu-Quan; Xue, Hui; Long, Jin-Hua; Wang, Yun; Jia, Yi; Qiu, Wei; Zhou, Jing; Wen, Zong-Yao; Yao, Wei-Juan; Zeng, Zhu

2016-01-01

Dendritic cells (DCs), the most potent antigen-presenting cells, play a central role in the initiation, regulation, and maintenance of the immune responses. Vascular endothelial growth factor (VEGF) is one of the important cytokines in the tumor microenvironment (TME) and can inhibit the differentiation and functional maturation of DCs. To elucidate the potential mechanisms of DC dysfunction induced by VEGF, the effects of VEGF on the biophysical characteristics and motility of human mature DCs (mDCs) were investigated. The results showed that VEGF had a negative influence on the biophysical properties, including electrophoretic mobility, osmotic fragility, viscoelasticity, and transmigration. Further cytoskeleton structure analysis by confocal microscope and gene expression profile analyses by gene microarray and real-time PCR indicated that the abnormal remodeling of F-actin cytoskeleton may be the main reason for the deterioration of biophysical properties, motility, and stimulatory capability of VEGF-treated mDCs. This is significant for understanding the biological behavior of DCs and the immune escape mechanism of tumors. Simultaneously, the therapeutic efficacies may be improved by blocking the signaling pathway of VEGF in an appropriate manner before the deployment of DC-based vaccinations against tumors. PMID:27809226

Current status and applications of somatic cell nuclear transfer in dogs.

PubMed

Jang, Goo; Kim, Min Kyu; Lee, Byeong Chun

2010-11-01

Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications. Copyright © 2010 Elsevier Inc. All rights reserved.

New Hippocampal Neurons Mature Rapidly in Response to Ketamine But Are Not Required for Its Acute Antidepressant Effects on Neophagia in Rats123

PubMed Central

Soumier, Amelie; Carter, Rayna M.; Schoenfeld, Timothy J.

2016-01-01

Abstract Virtually all antidepressant agents increase the birth of granule neurons in the adult dentate gyrus in rodents, providing a key basis for the neurogenesis hypothesis of antidepressant action. The novel antidepressant ketamine, however, shows antidepressant activity in humans within hours, far too rapid for a mechanism involving neuronal birth. Ketamine could potentially act more rapidly by enhancing maturation of new neurons born weeks earlier. To test this possibility, we assessed the effects of S-ketamine (S-(+)-ketamine hydrochloride) injection on maturation, as well as birth and survival, of new dentate gyrus granule neurons in rats, using the immediate-early gene zif268, proliferating cell nuclear antigen, and BrdU, respectively. We show that S-ketamine has rapid effects on new neurons, increasing the proportion of functionally mature young granule neurons within 2 h. A single injection of S-ketamine also increased cell proliferation and functional maturation, and decreased depressive-like behavior, for at least 4 weeks in rats treated with long-term corticosterone administration (a depression model) and controls. However, the behavioral effects of S-ketamine on neophagia were unaffected by elimination of adult neurogenesis. Together, these results indicate that ketamine has surprisingly rapid and long-lasting effects on the recruitment of young neurons into hippocampal networks, but that ketamine has antidepressant-like effects that are independent of adult neurogenesis. PMID:27066531

No Amelioration of Uromodulin Maturation and Trafficking Defect by Sodium 4-Phenylbutyrate in Vivo

PubMed Central

Kemter, Elisabeth; Sklenak, Stefanie; Rathkolb, Birgit; Hrabě de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard; Wanke, Ruediger

2014-01-01

Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium 4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as a potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IκB kinase α/β, an indirect activator of NF-κB. Furthermore, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52, and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention. PMID:24567330

Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Saitou, Mitinori

2013-08-01

Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

Mesenchymal Stem Cells Modulate Differentiation of Myeloid Progenitor Cells During Inflammation.

PubMed

Amouzegar, Afsaneh; Mittal, Sharad K; Sahu, Anuradha; Sahu, Srikant K; Chauhan, Sunil K

2017-06-01

Mesenchymal stem cells (MSCs) possess distinct immunomodulatory properties and have tremendous potential for use in therapeutic applications in various inflammatory diseases. MSCs have been shown to regulate pathogenic functions of mature myeloid inflammatory cells, such as macrophages and neutrophils. Intriguingly, the capacity of MSCs to modulate differentiation of myeloid progenitors (MPs) to mature inflammatory cells remains unknown to date. Here, we report the novel finding that MSCs inhibit the expression of differentiation markers on MPs under inflammatory conditions. We demonstrate that the inhibitory effect of MSCs is dependent on direct cell-cell contact and that this intercellular contact is mediated through interaction of CD200 expressed by MSCs and CD200R1 expressed by MPs. Furthermore, using an injury model of sterile inflammation, we show that MSCs promote MP frequencies and suppress infiltration of inflammatory cells in the inflamed tissue. We also find that downregulation of CD200 in MSCs correlates with abrogation of their immunoregulatory function. Collectively, our study provides unequivocal evidence that MSCs inhibit differentiation of MPs in the inflammatory environment via CD200-CD200R1 interaction. Stem Cells 2017;35:1532-1541. © 2017 AlphaMed Press.

Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix—A Comparative Analysis of Bioartificial Liver Microenvironments

PubMed Central

Wang, Bo; Jakus, Adam E.; Baptista, Pedro M.; Soker, Shay; Soto-Gutierrez, Alejandro; Abecassis, Michael M.; Shah, Ramille N.

2016-01-01

Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-l-lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a “deconstructed” control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, α-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery. Significance Through the use of novel technology to develop three-dimensional (3D) scaffolds, the present study demonstrated that hepatocyte-like cells derived via induced pluripotent stem cell (iPSC) technology mature on 3D extracellular matrix scaffolds as a result of 3D matrix structure and scaffold biology. The result is an improved hepatic phenotype with increased synthetic and catalytic potency, an improvement on the blunted phenotype of iPSC-derived hepatocytes, a critical limitation of iPSC technology. These findings provide insight into the influence of 3D microenvironments on the viability, proliferation, and function of iPSC hepatocytes to yield a more mature population of cells for cell toxicity studies and disease modeling. PMID:27421950

Systematic characterization of maturation time of fluorescent proteins in living cells

PubMed Central

Balleza, Enrique; Kim, J. Mark; Cluzel, Philippe

2017-01-01

Slow maturation time of fluorescent proteins limits accurate measurement of rapid gene expression dynamics and effectively reduces fluorescence signal in growing cells. We used high-precision time-lapse microscopy to characterize, at two different temperatures in E. coli, the maturation kinetics of 50 FPs that span the visible spectrum. We identified fast-maturing FPs that yield the highest signal-to-noise ratio and temporal resolution in individual growing cells. PMID:29320486

Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells.

PubMed

Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J; Tarle, Susan A; Kaigler, Darnell

2014-04-01

In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential, which could make them a very attractive cell population for utilization in regenerative cell therapies.

Expression of a Broad Array of Negative Costimulatory Molecules and Blimp-1 in T Cells following Priming by HIV-1 Pulsed Dendritic Cells

PubMed Central

Shankar, Esaki Muthu; Che, Karlhans Fru; Messmer, Davorka; Lifson, Jeffrey D; Larsson, Marie

2011-01-01

Accumulating evidence indicates that immune impairment in persistent viral infections could lead to T-cell exhaustion. To evaluate the potential contribution of induction of negative costimulatory molecules to impaired T-cell responses, we primed naïve T cells with mature monocyte-derived dendritic cells (MDDCs) pulsed with HIV-1 in vitro. We used quantitative real-time polymerase chain reaction and flow cytometry, respectively, to compare the gene and surface-protein expression profiles of naïve T cells primed with HIV-pulsed or mock-pulsed DCs. We detected elevated expressions of negative costimulatory molecules, including lymphocyte activation gene-3 (LAG-3), CD160, cytolytic T-lymphocyte antigen-4 (CTLA-4), T-cell immunoglobulin mucin-containing domain-3 (TIM-3), programmed death-1 (PD-1) and TRAIL (tumor necrosis-factor–related apoptosis-inducing ligand) in T cells primed by HIV-pulsed DCs. The PD-1+ T-cell population also coexpressed TIM-3, LAG-3, and CTLA-4. Interestingly, we also found an increase in gene expression of the transcriptional repressors Blimp-1 (B-lymphocyte–induced maturation protein-1) and Foxp3 (forkhead transcription factor) in T-cells primed by HIV-pulsed DCs; Blimp-1 expression was directly proportional to the expression of the negative costimulatory molecules. Furthermore, levels of the effector cytokines interleukin-2, tumor necrosis factor-α and interferon-γ, and perforin and granzyme B were decreased in T-cell populations primed by HIV-pulsed DCs. In conclusion, in vitro priming of naïve T-cells with HIV-pulsed DC leads to expansion of T cells with coexpression of a broad array of negative costimulatory molecules and Blimp-1, with potential deleterious consequences for T-cell responses. PMID:21103670

A decay of gap junctions associated with ganglion cell differentiation during retinal regeneration of the adult newt.

PubMed

Oi, Hanako; Chiba, Chikafumi; Saito, Takehiko

2003-12-01

Changes in the gap junctional coupling and maturation of voltage-activated Na(+) currents during regeneration of newt retinas were examined by whole-cell patch-clamping in slice preparations. Progenitor cells in regenerating retinas did not exhibit Na(+) currents but showed prominent electrical and tracer couplings. Cells identified by LY-fills were typically slender. Na(+) currents were detected in premature ganglion cells with round somata in the 'intermediate-II' regenerating retina. No electrical and tracer couplings were observed between these cells. Mature ganglion cells did not exhibit electrical coupling, but showed tracer coupling. On average, the maximum Na(+) current amplitude recorded from premature ganglion cells was roughly 2.5-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current was nearly 11 mV more positive than that of mature cells. We provide morphological and physiological evidence showing that loss of gap junctions between progenitor cells is associated with ganglion cell differentiation during retinal regeneration and that new gap junctions are recreated between mature ganglion cells. Also we provide evidence suggesting that the loss of gap junctions correlates with the appearance of voltage-activated Na(+) currents in ganglion cells.

Regulatory role of periodontal ligament fibroblasts for innate immune cell function and differentiation.

PubMed

Konermann, Anna; Stabenow, Dirk; Knolle, Percy A; Held, Stefanie A E; Deschner, James; Jäger, Andreas

2012-10-01

Innate immunity is crucial for an effective host defense against pathogenic microorganisms in periodontal tissues. As periodontal ligament (PDL) cells synthesize immunomodulatory cytokines, the aim of this in vitro study was to investigate whether these cells can interact with innate immune cells. Resting and inflammatory primed (IL-1β, TNF-α, HMGB1) human PDL cells were co-cultured with human monocyte-derived dendritic cells or macrophages. Migration, phenotypic maturation and modulation of phagocytosis of Porphyromonas gingivalis by immune cells were investigated upon co-culture with PDL cells and/or their released soluble factors. PDL cells interacted with immune cells under both non-inflammatory and inflammatory conditions. Immune cell migration was significantly enhanced by co-culture with PDL cells, which also affected their phenotypic maturation both through cell-cell contact and through released soluble mediators. The dendritic cell maturation markers CD83 and CD86 were upregulated as much as both 'alternatively activated' M2 macrophage maturation markers CD23 and CD163. In contrast, the 'classically activated' M1 macrophage maturation marker CD64 was downregulated. Finally, PDL cells significantly enhanced the phagocytosis of Porphyromonas gingivalis by immune cells. Our experiments revealed that PDL cells are not only structural elements of the periodontium, but actively influence immune responses by interaction with innate immune cells.

Intratubular germ cell neoplasia of the human testis: heterogeneous protein expression and relation to invasive potential

PubMed Central

Mitchell, Rod T; Camacho-Moll, Maria; Macdonald, Joni; Anderson, Richard A; Kelnar, Christopher JH; O’Donnell, Marie; Sharpe, Richard M; Smith, Lee B; Grigor, Ken M; Wallace, W Hamish B; Stoop, Hans; Wolffenbuttel, Katja P; Donat, Roland

2014-01-01

Testicular germ cell cancer develops from pre-malignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4+/ MAGEA4−) into pre-spermatogonia (OCT4−/MAGEA4+). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesised that cells expressing an immature (OCT4+/MAGEA4−) germ cell profile would exhibit an increased proliferation rate compared to those with a mature profile (OCT4+/ MAGEA4+). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with pre-invasive disease, seminoma and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4+/MAGEA4- cells showed a significantly increased rate of proliferation compared with the OCT4+/MAGEA4+ population (12.8 v 3.4%, p<0.0001) irrespective of histological tumour type, reflected in the predominance of OCT4+/MAGEA4− cells in the invasive tumour component. Surprisingly, OCT4+/MAGEA4− cells in patients with pre-invasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 v 10.2 v 7.2%, p<0.05 respectively). In conclusion, this study has demonstrated that OCT4+/MAGEA4− cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas. PMID:24457464

Cannabinoids induce incomplete maturation of cultured human leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murison, G.; Chubb, C.B.H.; Maeda, S.

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. Themore » THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.« less

Direct Hydrogel Encapsulation of Pluripotent Stem Cells Enables Ontomimetic Differentiation and Growth of Engineered Human Heart Tissues

PubMed Central

Kerscher, Petra; Turnbull, Irene C; Hodge, Alexander J; Kim, Joonyul; Seliktar, Dror; Easley, Christopher J; Costa, Kevin D; Lipke, Elizabeth A

2016-01-01

Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. Here we demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue. PMID:26826618

Adoptive Cell Therapy of Induced Regulatory T Cells Expanded by Tolerogenic Dendritic Cells on Murine Autoimmune Arthritis.

PubMed

Yang, Jie; Liu, Lidong; Yang, Yiming; Kong, Ning; Jiang, Xueyu; Sun, Juan; Xie, Rufeng

2017-01-01

Tolerogenic dendritic cells (tDCs) can expand TGF- β -induced regulatory T cells (iTregs); however, the therapeutic utility of these expanded iTregs in autoimmune diseases remains unknown. We sought to determine the properties of iTregs expanded by mature tolerogenic dendritic cells (iTreg mtDC ) in vitro and explore their potential to ameliorate collagen-induced arthritis (CIA) in a mouse model. After induction by TGF- β and expansion by mature tDCs (mtDCs), the phenotype and proliferation of iTreg mtDC were assessed by flow cytometry. The ability of iTregs and iTreg mtDC to inhibit CD4 + T cell proliferation and suppress Th17 cell differentiation was compared. Following adoptive transfer of iTregs and iTreg mtDC to mice with CIA, the clinical and histopathologic scores, serum levels of IFN- γ , TNF- α , IL-17, IL-6, IL-10, TGF- β and anti-CII antibodies, and the distribution of the CD4 + Th subset were assessed. Compared with iTregs, iTreg mtDC expressed higher levels of Foxp3 and suppressed CD4 + T cell proliferation and Th17 cell differentiation to a greater extent. In vivo, iTreg mtDC reduced the severity and progression of CIA more significantly than iTregs, which was associated with a modulated inflammatory cytokine profile, reduced anti-CII IgG levels, and polarized Treg/Th17 balance. This study highlights the potential therapeutic utility of iTreg mtDC in autoimmune arthritis and should facilitate the future design of iTreg immunotherapeutic strategies.

Adoptive Cell Therapy of Induced Regulatory T Cells Expanded by Tolerogenic Dendritic Cells on Murine Autoimmune Arthritis

PubMed Central

Liu, Lidong; Kong, Ning; Jiang, Xueyu; Sun, Juan; Xie, Rufeng

2017-01-01

Objective Tolerogenic dendritic cells (tDCs) can expand TGF-β-induced regulatory T cells (iTregs); however, the therapeutic utility of these expanded iTregs in autoimmune diseases remains unknown. We sought to determine the properties of iTregs expanded by mature tolerogenic dendritic cells (iTregmtDC) in vitro and explore their potential to ameliorate collagen-induced arthritis (CIA) in a mouse model. Methods After induction by TGF-β and expansion by mature tDCs (mtDCs), the phenotype and proliferation of iTregmtDC were assessed by flow cytometry. The ability of iTregs and iTregmtDC to inhibit CD4+ T cell proliferation and suppress Th17 cell differentiation was compared. Following adoptive transfer of iTregs and iTregmtDC to mice with CIA, the clinical and histopathologic scores, serum levels of IFN-γ, TNF-α, IL-17, IL-6, IL-10, TGF-β and anti-CII antibodies, and the distribution of the CD4+ Th subset were assessed. Results Compared with iTregs, iTregmtDC expressed higher levels of Foxp3 and suppressed CD4+ T cell proliferation and Th17 cell differentiation to a greater extent. In vivo, iTregmtDC reduced the severity and progression of CIA more significantly than iTregs, which was associated with a modulated inflammatory cytokine profile, reduced anti-CII IgG levels, and polarized Treg/Th17 balance. Conclusion This study highlights the potential therapeutic utility of iTregmtDC in autoimmune arthritis and should facilitate the future design of iTreg immunotherapeutic strategies. PMID:28702462

Recent thymic emigrants and mature naive T cells exhibit differential DNA methylation at key cytokine loci.

PubMed

Berkley, Amy M; Hendricks, Deborah W; Simmons, Kalynn B; Fink, Pamela J

2013-06-15

Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naive peripheral T cell pool. We show in this study that the Il2 and Il4 promoter regions of naive CD4(+) RTEs are characterized by site-specific hypermethylation compared with those of both mature naive (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared with MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for postthymic maturation.

Fibroblast Growth Factor 10 Enhances the Developmental Efficiency of Somatic Cell Nuclear Transfer Embryos by Accelerating the Kinetics of Cleavage During In Vitro Maturation.

PubMed

Son, Yeo-Jin; Lee, Seung-Eun; Park, Yun-Gwi; Jeong, Sang-Gi; Shin, Min-Young; Kim, Eun-Young; Park, Se-Pill

2018-06-01

Somatic cell nuclear transfer (SCNT) is required for the generation of transgenic animals as disease models. During the in vitro development of SCNT embryos, the quality of matured oocytes is one of the major factors regulating the developmental potential of embryos. Time-lapse monitoring systems are new tools that assess the developmental capacity of embryos for use in embryo transfer. In this study, we investigated the effect of fibroblast growth factor 10 (FGF 10) on the developmental potential of SCNT embryos. After the in vitro maturation (IVM) of oocytes in IVM medium containing 10 ng/mL FGF 10 (10 F), the polar body extrusion rate was significantly higher than in the control. However, there was no difference in the percentage of fused embryos between the groups. The cleavage and blastocyst formation rates of embryos were significantly increased in the 10 F compared with the control. In addition, the total cell number was higher and the apoptotic index was lower in the 10 F than control at day 7. The messenger RNA (mRNA) expression of genes involved in apoptosis (baculoviral inhibitor of apoptosis repeat containing 5 [BIRC5] and caspase 3 [CASP3]) and development (octamer-binding transcription factor 4 [POU5F1] and sex determining region Y box 2 [SOX2]) increased after 10 F treatment. Furthermore, the kinetics of the first cleavage was faster and the percentage of embryos at cell block was significantly lower in the 10 F group than in the control. These results demonstrate that exposure of oocytes to FGF 10 during IVM promotes developmental competence.

Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

PubMed Central

Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

2014-01-01

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

PubMed

Sudiman, Jaqueline; Sutton-McDowall, Melanie L; Ritter, Lesley J; White, Melissa A; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

2014-01-01

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

PubMed

Bojnordi, Maryam Nazm; Azizi, Hossein; Skutella, Thomas; Movahedin, Mansoureh; Pourabdolhossein, Fereshteh; Shojaei, Amir; Hamidabadi, Hatef Ghasemi

2017-09-01

Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N 2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B 27 , N 2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and β-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

Distinct Physiological Maturation of Parvalbumin-Positive Neuron Subtypes in Mouse Prefrontal Cortex.

PubMed

Miyamae, Takeaki; Chen, Kehui; Lewis, David A; Gonzalez-Burgos, Guillermo

2017-05-10

Parvalbumin-positive (PV + ) neurons control the timing of pyramidal cell output in cortical neuron networks. In the prefrontal cortex (PFC), PV + neuron activity is involved in cognitive function, suggesting that PV + neuron maturation is critical for cognitive development. The two major PV + neuron subtypes found in the PFC, chandelier cells (ChCs) and basket cells (BCs), are thought to play different roles in cortical circuits, but the trajectories of their physiological maturation have not been compared. Using two separate mouse lines, we found that in the mature PFC, both ChCs and BCs are abundant in superficial layer 2, but only BCs are present in deeper laminar locations. This distinctive laminar distribution was observed by postnatal day 12 (P12), when we first identified ChCs by the presence of axon cartridges. Electrophysiology analysis of excitatory synapse development, starting at P12, showed that excitatory drive remains low throughout development in ChCs, but increases rapidly before puberty in BCs, with an earlier time course in deeper-layer BCs. Consistent with a role of excitatory synaptic drive in the maturation of PV + neuron firing properties, the fast-spiking phenotype showed different maturation trajectories between ChCs and BCs, and between superficial versus deep-layer BCs. ChC and BC maturation was nearly completed, via different trajectories, before the onset of puberty. These findings suggest that ChC and BC maturation may contribute differentially to the emergence of cognitive function, primarily during prepubertal development. SIGNIFICANCE STATEMENT Parvalbumin-positive (PV + ) neurons tightly control pyramidal cell output. Thus PV + neuron maturation in the prefrontal cortex (PFC) is crucial for cognitive development. However, the relative physiological maturation of the two major subtypes of PV + neurons, chandelier cells (ChCs) and basket cells (BCs), has not been determined. We assessed the maturation of ChCs and BCs in different layers of the mouse PFC, and found that, from early postnatal age, ChCs and BCs differ in laminar location. Excitatory synapses and fast-spiking properties matured before the onset of puberty in both cell types, but following cell type-specific developmental trajectories. Hence, the physiological maturation of ChCs and BCs may contribute to the emergence of cognitive function differentially, and predominantly during prepubertal development. Copyright © 2017 the authors 0270-6474/17/374883-20$15.00/0.

Adenocarcinoma arising from intracranial recurrent mature teratoma and featuring mutated KRAS and wild-type BRAF genes.

PubMed

Kim, Eun Soo; Kwon, Mi Jung; Song, Joon Ho; Kim, Dong Hoon; Park, Hye-Rim

2015-02-01

Malignant transformation or recurrence of intracranial mature teratoma is an extremely rare occurrence, compared to the usual ovarian counterpart. Previously, yolk sac tumor elements have been considered to be selective progenitors of enteric-type adenocarcinoma arising from intracranial germ cell tumors. However, the present case demonstrates the occurrence of enteric-type adenocarcinoma in recurrent intracranial mature cystic teratoma 12 years after gross total removal, a case of which has not previously been documented in the literature. The 11.5-cm long, dura mater-based tumor on the right fronto-temporal lobe displaced the brain; however, the patient had no neurologic symptoms or discomfort other than pus-like discharge on the scalp. Microscopic examinations revealed a small focus of adenocarcinoma and dysplastic colonic mucosa in the mature cystic teratoma. No immature elements were seen. The cystic wall was almost denuded and showed an exuberant xanthogranulomatous reaction with foreign-body type giant cells engulfing keratin materials and cholesterol clefts, suggesting that chronic inflammation due to repeated cyst wall rupture and the previous resection may contribute to malignant transformation. The adenocarcinoma showed strong immunohistochemical expression of CK20 and p53, but CK7 in patches. The molecular profile of the adenocarcinoma showed a mutation in KRAS and wild-type BRAF, which might be associated with malignant transformation of intracranial mature teratomas. In conclusion, the intracranial mature teratomas should require long-term follow-up, and clinicians, radiologists and pathologists should be aware of the potential for malignant progression of recurrent intracranial mature cystic teratoma despite gross total resection and no neurologic symptoms. © 2014 Japanese Society of Neuropathology.

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

PubMed

Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

2016-04-01

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P < 0.05), while the results for the cumulus-oocyte complex groups were similar between the experimental groups and control groups. The results of this study indicated that platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling.

PubMed

Penafuerte, Claudia; Feldhammer, Matthew; Mills, John R; Vinette, Valerie; Pike, Kelly A; Hall, Anita; Migon, Eva; Karsenty, Gerard; Pelletier, Jerry; Zogopoulos, George; Tremblay, Michel L

2017-01-01

PTP1B and TC-PTP are highly related protein-tyrosine phosphatases (PTPs) that regulate the JAK/STAT signaling cascade essential for cytokine-receptor activation in immune cells. Here, we describe a novel immunotherapy approach whereby monocyte-derived dendritic cell (moDC) function is enhanced by modulating the enzymatic activities of PTP1B and TC-PTP. To downregulate or delete the activity/expression of these PTPs, we generated mice with PTP-specific deletions in the dendritic cell compartment or used PTP1B and TC-PTP specific inhibitor. While total ablation of PTP1B or TC-PTP expression leads to tolerogenic DCs via STAT3 hyperactivation, downregulation of either phosphatase remarkably shifts the balance toward an immunogenic DC phenotype due to hyperactivation of STAT4, STAT1 and Src kinase. The resulting increase in IL-12 and IFNγ production subsequently amplifies the IL-12/STAT4/IFNγ/STAT1/IL-12 positive autocrine loop and enhances the therapeutic potential of mature moDCs in tumor-bearing mice. Furthermore, pharmacological inhibition of both PTPs improves the maturation of defective moDCs derived from pancreatic cancer (PaC) patients. Our study provides a new advance in the use of DC-based cancer immunotherapy that is complementary to current cancer therapeutics.

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation.

PubMed

Cui, Xiuji; Luckenbaugh, Laurie; Bruss, Volker; Hu, Jianming

2015-10-01

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Potential for Aedes albopictus and Ochlerotatus j. japonicus to Change the Field Ecology of Arboviruses of Human Health Importance in the Mid-Atlantic Region of the United States

DTIC Science & Technology

2001-10-16

in the midgut epithelial cells. Step 3 is release 17 of virus from the midgut epithelial cell into the hemolymph. Step 4 is infection of the...proteins, maturation, and budding. During attachment, the viral attachment proteins on the surface of the virion bind to receptors on the microvillar...membrane of the mosquito s midgut . Penetration refers to the process through which the virions enter the midgut cells; arboviruses enter cell

Reinterpreting recent thymic emigrant function: defective or adaptive?

PubMed

Cunningham, Cody A; Helm, Eric Y; Fink, Pamela J

2018-04-01

Recent thymic emigrants (RTEs) are those peripheral T cells that have most recently completed thymic development and egress. Over the past decade, significant advances have been made in understanding the cell-extrinsic and cell-intrinsic requirements for RTE maturation to mature naïve (MN) T cells and in detailing the functional differences that characterize these two T cell populations. Much of this work has suggested that RTEs are hypo-functional versions of more mature T cells. However, recent evidence has indicated that rather than being defective T cells, RTEs are exquisitely adapted to their cellular niche. In this review, we argue that RTEs are not flawed mature T cells but are adapted to fill an underpopulated T cell compartment, while maintaining self tolerance and possessing the capacity to mount robust immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Clonal mature adipocyte production of proliferative-competent daughter cells requires lipid export prior to cell division

USDA-ARS?s Scientific Manuscript database

Numerous in vitro observations have been published to show that mature adipocytes may resume proliferation and begin to populate the adipofibroblast fraction or form other cell types. In the present study, we evaluated clonal cultures of mature pig-derived adipocytes as they began to reestablish the...

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

PubMed Central

Kobari, Ladan; Yates, Frank; Oudrhiri, Noufissa; Francina, Alain; Kiger, Laurent; Mazurier, Christelle; Rouzbeh, Shaghayegh; El-Nemer, Wassim; Hebert, Nicolas; Giarratana, Marie-Catherine; François, Sabine; Chapel, Alain; Lapillonne, Hélène; Luton, Dominique; Bennaceur-Griscelli, Annelise; Douay, Luc

2012-01-01

Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. PMID:22733021

Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

PubMed

Huber, Birgit; Czaja, Alina Maria; Kluger, Petra Juliane

2016-05-01

The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used. © 2016 International Federation for Cell Biology.

Stem cells and female reproduction.

PubMed

Du, Hongling; Taylor, Hugh S

2009-02-01

Several recent findings in stem cell biology have resulted in new opportunities for the treatment of reproductive disease. Endometrial regeneration can be driven by bone marrow derived stem cells. This finding has potential implications for the treatment of uterine disorders. It also supports a new theory for the etiology of endometriosis. The ovaries have been shown to contain stem cells that form oocytes in adults and can be cultured in vitro to develop mature oocytes. Stem cells from the fetus have been demonstrated to lead to microchimerism in the mother and implicated in several maternal diseases. Additionally the placenta may be another source of hematopoietic stem cell. Finally endometrial derived stem cells have been demonstrated to differentiate into non-reproductive tissues. While we are just beginning to understand stem cells and many key questions remain, the potential advantages of stem cells in reproductive biology and medicine are apparent.

Circulating B cells in type 1 diabetics exhibit fewer maturation-associated phenotypes.

PubMed

Hanley, Patrick; Sutter, Jennifer A; Goodman, Noah G; Du, Yangzhu; Sekiguchi, Debora R; Meng, Wenzhao; Rickels, Michael R; Naji, Ali; Luning Prak, Eline T

2017-10-01

Although autoantibodies have been used for decades as diagnostic and prognostic markers in type 1 diabetes (T1D), further analysis of developmental abnormalities in B cells could reveal tolerance checkpoint defects that could improve individualized therapy. To evaluate B cell developmental progression in T1D, immunophenotyping was used to classify circulating B cells into transitional, mature naïve, mature activated, and resting memory subsets. Then each subset was analyzed for the expression of additional maturation-associated markers. While the frequencies of B cell subsets did not differ significantly between patients and controls, some T1D subjects exhibited reduced proportions of B cells that expressed transmembrane activator and CAML interactor (TACI) and Fas receptor (FasR). Furthermore, some T1D subjects had B cell subsets with lower frequencies of class switching. These results suggest circulating B cells exhibit variable maturation phenotypes in T1D. These phenotypic variations may correlate with differences in B cell selection in individual T1D patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells.

PubMed

Chen, Chen; Soto-Gutierrez, Alejandro; Baptista, Pedro M; Spee, Bart

2018-04-01

The incidence of liver disease is increasing globally. The only curative therapy for severe end-stage liver disease, liver transplantation, is limited by the shortage of organ donors. In vitro models of liver physiology have been developed and new technologies and approaches are progressing rapidly. Stem cells might be used as a source of liver tissue for development of models, therapies, and tissue-engineering applications. However, we have been unable to generate and maintain stable and mature adult liver cells ex vivo. We review factors that promote hepatocyte differentiation and maturation, including growth factors, transcription factors, microRNAs, small molecules, and the microenvironment. We discuss how the hepatic circulation, microbiome, and nutrition affect liver function, and the criteria for considering cells derived from stem cells to be fully mature hepatocytes. We explain the challenges to cell transplantation and consider future technologies for use in hepatic stem cell maturation, including 3-dimensional biofabrication and genome modification. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Kuang, A.; Musgrave, M. E.

1996-01-01

Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation in Arabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultrastructure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyosomes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.

Mature aggressive B-cell lymphoma across age groups - molecular advances and therapeutic implications.

PubMed

Lange, Jonas; Lenz, Georg; Burkhardt, Birgit

2017-02-01

Mature B-cell lymphoma represents the most common type of Non-Hodgkin lymphoma, and different subtypes prevail at different patient ages. Areas covered: We review recent data on differences and commonalities in mature B-cell lymphoma occurring in adult and pediatric patients, with a special emphasis on molecular advances and therapeutic implications. To this end, we will discuss knowledge on diffuse large B-cell lymphoma and Burkitt lymphoma/leukemia, which are the most frequent subtypes in adult and pediatric patients, respectively, and on primary mediastinal B-cell lymphoma, which is a subtype of mature B-cell lymphoma occurring mainly in adolescents and young adults with a female predominance. Expert commentary: Molecular profiling has revealed molecular alterations that can be used to further classify the subtypes of mature B-cell lymphoma. These new subgroups frequently respond differentially to targeted therapeutic strategies. Future clinical trials utilizing new drugs will address this issue by combining clinical data and response assessment with a molecular workup of the corresponding lymphomas.

Expansion and hepatic differentiation of rat multipotent adult progenitor cells in microcarrier suspension culture.

PubMed

Park, Y; Subramanian, K; Verfaillie, C M; Hu, W S

2010-10-01

Many potential applications of stem cells require large quantities of cells, especially those involving large organs such as the liver. For such applications, a scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiation competent or differentiated cells. We employed a microcarrier culture system for the expansion of undifferentiated rat multipotent adult progenitor cells (rMAPC) as well as for directed differentiation of these cells to hepatocyte-like cells. During the 4-day expansion culture, cell concentration increased by 85-fold while expression level of pluripotency markers were maintained, as well as the MAPC differentiation potential. Directed differentiation into hepatocyte-like cells on the microcarriers themselves gave comparable results as observed with cells cultured in static cultures. The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Microcarrier culture thus offers the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment. Copyright © 2010 Elsevier B.V. All rights reserved.

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell.

PubMed

Jelicks, L A; Weaver, J; Pollack, S; Gupta, R K

1989-08-15

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki

2014-05-15

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether directmore » cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.« less

Analysis of Ly49 gene transcripts in mature NK cells supports a role for the Pro1 element in gene activation, not gene expression.

PubMed

McCullen, M V; Li, H; Cam, M; Sen, S K; McVicar, D W; Anderson, S K

2016-09-01

The variegated expression of murine Ly49 loci has been associated with the probabilistic behavior of an upstream promoter active in immature cells, the Pro1 element. However, recent data suggest that Pro1 may be active in mature natural killer (NK) cells and function as an enhancer element. To assess directly if Pro1 transcripts are present in mature Ly49-expressing NK cells, RNA-sequencing of the total transcript pool was performed on freshly isolated splenic NK cells sorted for expression of either Ly49G or Ly49I. No Pro1 transcripts were detected from the Ly49a, Ly49c or Ly49i genes in mature Ly49(+) NK cells that contained high levels of Pro2 transcripts. Low levels of Ly49g Pro1 transcripts were found in both Ly49G(+) and Ly49G(-) populations, consistent with the presence of a small population of mature NK cells undergoing Ly49g gene activation, as previously demonstrated by culture of splenic NK cells in interleukin-2. Ly49 gene reporter constructs containing Pro1 failed to show any enhancer activity of Pro1 on Pro2 in a mature Ly49-expressing cell line. Taken together, the results are consistent with Pro1 transcription having a role in gene activation in developing NK, and argue against a role for Pro1 in Ly49 gene transcription by mature NK cells.

Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

PubMed

Gounni, A S; Gregory, B; Nutku, E; Aris, F; Latifa, K; Minshall, E; North, J; Tavernier, J; Levit, R; Nicolaides, N; Robinson, D; Hamid, Q

2000-09-15

Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

NBCe1 (SLC4A4) a potential pH Regulator in Enamel Organ Cells during Enamel Development in the Mouse

PubMed Central

Jalali, R; Guo, J; Zandieh-Doulabi, B; Bervoets, TJM; Paine, ML; Boron, W; Parker, M; Bijvelds, MJC; Medina, JF; DenBesten, PK; Bronckers, ALJJ

2016-01-01

During formation of dental enamel maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by co-transporting HCO3− with Na+. Mutation in SLC4A4 (coding for the Na+ bicarbonate co-transporter NBCe1) induces developmental defects in human and murine enamel. We hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in mouse wild-type dental epithelium and examined the effect of NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice weak to moderate immunostaining for NBCe1 with antibodies that recognize isoforms A/B/D/E and isoform C was seen in ameloblasts in secretory stage, no or very low staining in early maturation-stage but moderately to high staining in late maturation-stage. The papillary layer showed the opposite pattern and immunostained prominently at early maturation-stage but gradually showed less staining at mid- and late maturation-stage. In NBCe1−/− mice ameloblasts were disorganized, the enamel thin and severely hypomineralized. Enamel organs of CFTR−/− and AE2a,b−/− mice (believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Our data show that expression of NBCe1 in ameloblast and papillary layer cell depends on developmental stage and possibly responds to pH changes. PMID:25012520

Cell water potential, osmotic potential, and turgor in the epidermis and mesophyll of transpiring leaves : Combined measurements with the cell pressure probe and nanoliter osmometer.

PubMed

Nonami, H; Schulze, E D

1989-01-01

Water potential, osmotic potential and turgor measurements obtained by using a cell pressure probe together with a nanoliter osmometer were compared with measurements obtained with an isopiestic psychrometer. Both types of measurements were conducted in the mature region of Tradescantia virginiana L. leaves under non-transpiring conditions in the dark, and gave similar values of all potentials. This finding indicates that the pressure probe and the osmometer provide accurate measurements of turgor, osmotic potentials and water potentials. Because the pressure probe does not require long equilibration times and can measure turgor of single cells in intact plants, the pressure probe together with the osmometer was used to determine in-situ cell water potentials, osmotic potentials and turgor of epidermal and mesophyll cells of transpiring leaves as functions of stomatal aperture and xylem water potential. When the xylem water potential was-0.1 MPa, the stomatal aperture was at its maximum, but turgor of both epidermal and mesophyll cells was relatively low. As the xylem water potential decreased, the stomatal aperture became gradually smaller, whereas turgor of both epidermal and mesophyll cells first increased and afterward decreased. Water potentials of the mesophyll cells were always lower than those of the epidermal cells. These findings indicate that evaporation of water is mainly occurring from mesophyll cells and that peristomatal transpiration could be less important than it has been proposed previously, although peristomatal transpiration may be directly related to regulation of turgor in the guard cells.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

CpG methylation patterns and decitabine treatment response in acute myeloid leukemia cells and normal hematopoietic precursors

PubMed Central

Negrotto, Soledad; Ng, Kwok Peng; Jankowska, Ania M.; Bodo, Juraj; Gopalan, Banu; Guinta, Kathryn; Mulloy, James C.; Hsi, Eric; Maciejewski, Jaroslaw; Saunthararajah, Yogen

2011-01-01

The DNA hypomethylating drug decitabine maintains normal hematopoietic stem cell (HSC) self-renewal but induces terminal differentiation in acute myeloid leukemia (AML) cells. The basis for these contrasting cell-fates, and for selective CpG hypomethylation by decitabine, is poorly understood. Promoter CpGs, with methylation measured by microarray, were classified by the direction of methylation change with normal myeloid maturation. In AML cells, the methylation pattern at maturation-responsive CpG suggested at least partial maturation. Consistent with partial maturation, in gene expression analyses, AML cells expressed high levels of the key lineage-specifying factor CEBPA, but relatively low levels of the key late-differentiation driver CEBPE. In methylation analysis by mass-spectrometry, CEBPE promoter CpG that are usually hypomethylated during granulocyte maturation were significantly hypermethylated in AML cells. Decitabine treatment induced cellular differentiation of AML cells, and the largest methylation decreases were at CpG that are hypomethylated with myeloid maturation, including CEBPE promoter CpG. In contrast, decitabine-treated normal HSC retained immature morphology, and methylation significantly decreased at CpG that are less methylated in immature cells. High expression of lineage-specifying factor and aberrant epigenetic repression of some key late-differentiation genes distinguishes AML cells from normal HSC and could explain the contrasting differentiation and methylation responses to decitabine. PMID:21836612

Preclinical evaluation of the immunomodulatory properties of cardiac adipose tissue progenitor cells using umbilical cord blood mesenchymal stem cells: a direct comparative study.

PubMed

Perea-Gil, Isaac; Monguió-Tortajada, Marta; Gálvez-Montón, Carolina; Bayes-Genis, Antoni; Borràs, Francesc E; Roura, Santiago

2015-01-01

Cell-based strategies to regenerate injured myocardial tissue have emerged over the past decade, but the optimum cell type is still under scrutiny. In this context, human adult epicardial fat surrounding the heart has been characterized as a reservoir of mesenchymal-like progenitor cells (cardiac ATDPCs) with potential clinical benefits. However, additional data on the possibility that these cells could trigger a deleterious immune response following implantation are needed. Thus, in the presented study, we took advantage of the well-established low immunogenicity of umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) to comparatively assess the immunomodulatory properties of cardiac ATDPCs in an in vitro allostimulatory assay using allogeneic mature monocyte-derived dendritic cells (MDDCs). Similar to UCBMSCs, increasing amounts of seeded cardiac ATDPCs suppressed the alloproliferation of T cells in a dose-dependent manner. Secretion of proinflammatory cytokines (IL6, TNFα, and IFNγ) was also specifically modulated by the different numbers of cardiac ATDPCs cocultured. In summary, we show that cardiac ATDPCs abrogate T cell alloproliferation upon stimulation with allogeneic mature MDDCs, suggesting that they could further regulate a possible harmful immune response in vivo. Additionally, UCBMSCs can be considered as valuable tools to preclinically predict the immunogenicity of prospective regenerative cells.

Mast cells contribute to scar formation during fetal wound healing.

PubMed

Wulff, Brian C; Parent, Allison E; Meleski, Melissa A; DiPietro, Luisa A; Schrementi, Megan E; Wilgus, Traci A

2012-02-01

Scar formation is a potentially detrimental process of tissue restoration in adults, affecting organ form and function. During fetal development, cutaneous wounds heal without inflammation or scarring at early stages of development; however, they begin to heal with significant inflammation and scarring as the skin becomes more mature. One possible cell type that could regulate the change from scarless to fibrotic healing is the mast cell. We show here that dermal mast cells in scarless wounds generated at embryonic day 15 (E15) are fewer in number, less mature, and do not degranulate in response to wounding as effectively as mast cells of fibrotic wounds made at embryonic day 18 (E18). Differences were also observed between cultured mast cells from E15 and E18 skin, with regard to degranulation and preformed cytokine levels. Injection of mast cell lysates into E15 wounds disrupted scarless healing, suggesting that mast cells interfere with scarless repair. Finally, wounds produced at E18, which normally heal with a scar, healed with significantly smaller scars in mast cell-deficient Kit(W/W-v) mice compared with Kit(+/+) littermates. Together, these data suggest that mast cells enhance scar formation, and that these cells may mediate the transition from scarless to fibrotic healing during fetal development.

Low dose naltrexone (LDN) enhances maturation of bone marrow dendritic cells (BMDCs).

PubMed

Meng, Jingjuan; Meng, Yiming; Plotnikoff, Nicholas P; Youkilis, Gene; Griffin, Noreen; Shan, Fengping

2013-12-01

It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrexone, a non-peptidic δ-opioid receptor selective antagonist and opioid receptors on BMDCs have been detected [1]. However, there is little prior data published on naltrexone and DCs. Therefore, we hypothesized that LDN could exert modulating effect on BMDCs. In present study, we studied influence of LDN on both phenotypic and functional maturation of BMDCs. Changes of BMDC post-treatment with LDN were evaluated using conventional light microscope and transmission electron microscopy (TEM); flow cytometry(FCM); cytochemistry; acid phosphatase activity(ACP) test; FITC-dextran bio-assay; mixed lymphocytes and enzyme-linked immunosorbent assay (ELISA). We have found that LDN enhances maturation of BMDCs as evidenced by 1) up-regulating the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs; 2) down-regulating the rates of pinocytosis and phagocytosis accompanied by the results of decreased ACP, and FITC-dextran bio-assay; 3) mounting potential of BMDCs to drive T cell; and 4) inducing secretion of higher levels of IL-12 and TNF-α. It is therefore concluded that LDN can efficiently promote the maturation of BMDCs via precise modulation inside and outside BMDCs. Our study has provided meaningful mode of action on the role of LDN in immunoregulation, and rationale on future application of LDN for enhancing host immunity in cancer therapy and potent use in the design of DC-based vaccines for a number of diseases.

CD10/neutral endopeptidase 24.11 regulates fetal lung growth and maturation in utero by potentiating endogenous bombesin-like peptides.

PubMed

King, K A; Hua, J; Torday, J S; Drazen, J M; Graham, S A; Shipp, M A; Sunday, M E

1993-05-01

Bombesin-like peptides (BLPs) are mitogens for bronchial epithelial cells and small cell lung carcinomas, and increase fetal lung growth and maturation in utero and in organ cultures. BLPs are hydrolyzed by the enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) which is expressed in bronchial epithelium and functions to inhibit BLP-mediated growth of small cell lung carcinomas. To determine whether CD10/NEP regulates peptide-mediated lung development, we administered a specific CD10/NEP inhibitor, SCH32615, to fetal mice in utero from gestational days e15-17. Fetal lung tissues were evaluated on e18 for: (a) growth using [3H]thymidine incorporation into nuclear DNA; and (b) maturation using: [3H]-choline incorporation into surfactant phospholipids, electron microscopy for type II pneumocytes, and Northern blot analyses for surfactant apoproteins A, B, and C. Inhibition of CD10/NEP stimulated [3H]thymidine incorporation into DNA (70% above baseline, P < 0.005), [3H]choline incorporation into surfactant phospholipids (38% above baseline, P < 0.005), increased numbers of type II pneumocytes (36% above baseline, P = 0.07), and fivefold higher surfactant protein A transcripts (P < 0.05). CD10/NEP-mediated effects were completely blocked by the specific bombesin receptor antagonist, [D-Phe12, Leu14]bombesin. These observations suggest that CD10/NEP regulates fetal lung growth and maturation mediated by endogenous BLPs.

CD10/neutral endopeptidase 24.11 regulates fetal lung growth and maturation in utero by potentiating endogenous bombesin-like peptides.

PubMed Central

King, K A; Hua, J; Torday, J S; Drazen, J M; Graham, S A; Shipp, M A; Sunday, M E

1993-01-01

Bombesin-like peptides (BLPs) are mitogens for bronchial epithelial cells and small cell lung carcinomas, and increase fetal lung growth and maturation in utero and in organ cultures. BLPs are hydrolyzed by the enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) which is expressed in bronchial epithelium and functions to inhibit BLP-mediated growth of small cell lung carcinomas. To determine whether CD10/NEP regulates peptide-mediated lung development, we administered a specific CD10/NEP inhibitor, SCH32615, to fetal mice in utero from gestational days e15-17. Fetal lung tissues were evaluated on e18 for: (a) growth using [3H]thymidine incorporation into nuclear DNA; and (b) maturation using: [3H]-choline incorporation into surfactant phospholipids, electron microscopy for type II pneumocytes, and Northern blot analyses for surfactant apoproteins A, B, and C. Inhibition of CD10/NEP stimulated [3H]thymidine incorporation into DNA (70% above baseline, P < 0.005), [3H]choline incorporation into surfactant phospholipids (38% above baseline, P < 0.005), increased numbers of type II pneumocytes (36% above baseline, P = 0.07), and fivefold higher surfactant protein A transcripts (P < 0.05). CD10/NEP-mediated effects were completely blocked by the specific bombesin receptor antagonist, [D-Phe12, Leu14]bombesin. These observations suggest that CD10/NEP regulates fetal lung growth and maturation mediated by endogenous BLPs. Images PMID:8486767

Stage-dependency of apoptosis and the blood-testis barrier in the dogfish shark (Squalus acanthias): cadmium-induced changes as assessed by vital fluorescence techniques.

PubMed

McClusky, Leon M

2006-09-01

Naturally occurring heavy metals and synthetic compounds are potentially harmful for testicular function but evidence linking heavy metal exposure to reduced semen parameters is inconclusive. Elucidation of the exact stage at which the toxicant interferes with spermatogenesis is difficult because the various germ cell stages may have different sensitivities to any given toxicant, germ cell development is influenced by supporting testicular somatic cells and the presence of inter-Sertoli cell tight junctions create a blood-testis barrier, sequestering meiotic and postmeiotic germ cells in a special microenvironment. Sharks such as Squalus acanthias provide a suitable model for studying aspects of vertebrate spermatogenosis because of their unique features: spermatogenesis takes place within spermatocysts and relies mainly on Sertoli cells for somatic cell support; spermatocysts are linearly arranged in a maturational order across the diameter of the elongated testis; spermatocysts containing germ cells at different stages of development are topographically separated, resulting in visible zonation in testicular cross sections. We have used the vital dye acridine orange and a novel fluorescence staining technique to study this model to determine (1) the efficacy of these methods in assays of apoptosis and blood-testis barrier function, (2) the sensitivity of the various spermatogonial generations in Squalus to cadmium (as an illustrative spermatotoxicant) and (3) the way that cadmium might affect more mature spermatogenic stages and other physiological processes in the testis. Our results show that cadmium targets early spermatogenic stages, where it specifically activates a cell death program in susceptible (mature) spermatogonial clones, and negatively affects blood-testis barrier function. Since other parameters are relatively unaffected by cadmium, the effects of this toxicant on apoptosis are presumably process-specific and not attributable to general toxicity.

Microglia in CNS development: Shaping the brain for the future.

PubMed

Mosser, Coralie-Anne; Baptista, Sofia; Arnoux, Isabelle; Audinat, Etienne

Microglial cells are the resident macrophages of the central nervous system (CNS) and are mainly known for their roles in neuropathologies. However, major recent developments have revealed that these immune cells actively interact with neurons in physiological conditions and can modulate the fate and functions of synapses. Originating from myeloid precursors born in the yolk sac, microglial cells invade the CNS during early embryonic development. As a consequence they can potentially influence neuronal proliferation, migration and differentiation as well as the formation and maturation of neuronal networks, thereby contributing to the entire shaping of the CNS. We review here recent evidence indicating that microglial cells are indeed involved in crucial steps of the CNS development, including neuronal survival and apoptosis, axonal growth, migration of neurons, pruning of supernumerary synapses and functional maturation of developing synapses. We also discuss current hypotheses proposing that diverting microglial cells of their physiological functions, by promoting the expression of an immune phenotype during development, may be central to neurodevelopmental disorders such as autism, schizophrenia and epilepsy. Copyright © 2017 Elsevier Ltd. All rights reserved.

ASSESSMENT OF PC12 CELL DIFFERENTIATION AND NEURITE GROWTH: A COMPARISON OF MORPHOLOGICAL AND NEUROCHEMICAL MEASURES.

EPA Science Inventory

In order to screen large numbers of chemicals for their potential to produce developmental neurotoxicity new, in vitro methods are needed. One approach is to develop methods based on the biologic processes which underlie brain development including the growth and maturation of ce...

Influence of In Vitro and In Vivo Oxygen Modulation on β Cell Differentiation From Human Embryonic Stem Cells

PubMed Central

Cechin, Sirlene; Álvarez-Cubela, Silvia; Giraldo, Jaime A.; Molano, Ruth D.; Villate, Susana; Ricordi, Camillo; Pileggi, Antonello; Inverardi, Luca

2014-01-01

The possibility of using human embryonic stem (hES) cell-derived β cells as an alternative to cadaveric islets for the treatment of type 1 diabetes is now widely acknowledged. However, current differentiation methods consistently fail to generate meaningful numbers of mature, functional β cells. In order to address this issue, we set out to explore the role of oxygen modulation in the maturation of pancreatic progenitor (PP) cells differentiated from hES cells. We have previously determined that oxygenation is a powerful driver of murine PP differentiation along the endocrine lineage of the pancreas. We hypothesized that targeting physiological oxygen partial pressure (pO2) levels seen in mature islets would help the differentiation of PP cells along the β-cell lineage. This hypothesis was tested both in vivo (by exposing PP-transplanted immunodeficient mice to a daily hyperbaric oxygen regimen) and in vitro (by allowing PP cells to mature in a perfluorocarbon-based culture device designed to carefully adjust pO2 to a desired range). Our results show that oxygen modulation does indeed contribute to enhanced maturation of PP cells, as evidenced by improved engraftment, segregation of α and β cells, body weight maintenance, and rate of diabetes reversal in vivo, and by elevated expression of pancreatic endocrine makers, β-cell differentiation yield, and insulin production in vitro. Our studies confirm the importance of oxygen modulation as a key variable to consider in the design of β-cell differentiation protocols and open the door to future strategies for the transplantation of fully mature β cells. PMID:24375542

Anti-ATLA (antibody to adult T-cell leukemia virus-associated antigen), highly positive in OKT4-positive mature T-cell malignancies.

PubMed

Tobinai, K; Nagai, M; Setoya, T; Shibata, T; Minato, K; Shimoyama, M

1983-01-01

Serum or plasma specimens from 252 patients with lymphoid malignancies were screened for reactivity with adult T-cell leukemia virus-associated antigen (ATLA), and the relationship between the immunologic phenotype of the tumor cells and ATLA reactivity was determined. Anti-ATLA antibodies were found in 24 (29.3%) of 82 patients with T-cell malignancy. In contrast, the antibodies were found in none of the 106 patients with B-cell malignancy and only rarely in patients with other lymphoid malignancies without blood transfusions. Among the patients with T-cell malignancy, anti-ATLA antibodies were found in 23 (45.1%) of the 51 patients with OKT4-positive mature T-cell (inducer/helper T-cell) malignancy, but in none of the patients with T-cell malignancy of pre-T, thymic T-cell or OKT8-positive mature T-cell (suppressor/cytotoxic T-cell) phenotype. Furthermore, among the OKT4-positive mature T-cell malignancies, the antibodies were found in 16 (84.2%) of 19 patients with ATL and in 5 (27.8%) of 18 patients with mature (peripheral) T-cell lymphoma, in none of four with typical T-chronic lymphocytic leukemia, in one of nine with mycosis fungoides and in the one patient with small-cell variant of Sézary's syndrome. These results suggest that anti-ATLA positive T-cell malignancies with OKT4-positive mature T-cell phenotype must be the same disease, because it is highly possible that they have the same etiology and the same cellular origin. In the atypical cases, it seems necessary to demonstrate monoclonal integration of proviral DNA of ATLV or HTLV into the tumor cells in order to establish the final diagnosis of ATL.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

PubMed Central

Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

2012-01-01

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction. PMID:22093611

Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

PubMed

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

*CHANGING PATTERN OF THE SUBCELLULAR DISTRIBUTION OF ERYTHROBLAST MACROPHAGE PROTEIN (EMP) DURING MACROPHAGE DIFFERENTIATION

PubMed Central

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp), mediates the attachment of erythroid cells to macrophages, and is required for normal differentiation of both cell lineages. In erythroid cells Emp is believed to be involved in nuclear extrusion however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data shows that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture show that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data shows that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation, and suggests that Emp may be involved in multiple cellular functions. PMID:17071116

Investigation of relationship between precursor of miRNA-944 and its mature form in lung squamous-cell carcinoma - the diagnostic value.

PubMed

Powrózek, Tomasz; Mlak, Radosław; Dziedzic, Marcin; Małecka-Massalska, Teresa; Sagan, Dariusz

2018-03-01

MicroRNA (miRNA) are attractive markers of lung cancer, due to their regulatory role in cell cycle. However, we know more about function of miRNA in cancer development, there is still little known about role of their precursors (primary miRNA; pri-miRNA) in tumorgenesis. In present study we investigated potential role of miRNA-944 and its precursor pri-miRNA-944 in development of squamous-cell lung cancer (SCC) and explored interdependence between miRNA precursor and its mature form. This is a first available literature report analyzing pri-miRNA as a cancer diagnostic marker. Expression of miRNA-944 and its precursor was analyzed in 58 fresh-frozen tissues of non-small cell lung cancer and corresponding adjacent non-cancerous tissues using qRT-PCR. Expression of pri-miRNA-944 was correlated with TP63 and miRNA-944. Using ROC analysis diagnostic accuracy of studied markers was evaluated. miRNA-944 and its precursor were significantly overexspressed in SCC compared to adenocarcinoma (AC) and non-cancerous tissue. pri-miRNA-944 strongly and positively correlated with TP63 (r = 0.739, p < 0.001) and with mature miRNA-944 expression (r = 0.691, p < 0.001). Also, TP63 expression significantly correlated with mature miRNA (r = 0.785, p < 0.001). Combined analysis of pri-miRNA-944 and mature miRNA-944 allowed to distinguish SCC tissue form AC with sensitivity of 93.3% and specificity of 100% (AUC = 0.978), and SCC from non-cancerous tissue with 92.9% sensitivity and 100% specificity (AUC = 0.992). We assumed that pri-miRNA-944 and miRNA-944 may be involved in early squamous-type differentiation of lung tumors. Moreover, analysis of both markers provided high diagnostic accuracy for SCC detection. Copyright © 2018 Elsevier GmbH. All rights reserved.

Effect of triiodothyronine on developmental competence of bovine oocytes.

PubMed

Costa, N N; Cordeiro, M S; Silva, T V G; Sastre, D; Santana, P P B; Sá, A L A; Sampaio, R V; Santos, S S D; Adona, P R; Miranda, M S; Ohashi, O M

2013-09-01

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Development of Mouse Embryonic Stem (ES) Cells: IV Differentiation to Mature T and B Lymphocytes after Implantation of Embryoid Bodies Into Nude Mice

PubMed Central

Mok, Hoyan

1995-01-01

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed. PMID:9700357

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

PubMed Central

Laugwitz, Karl-Ludwig; Moretti, Alessandra; Lam, Jason; Gruber, Peter; Chen, Yinhong; Woodard, Sarah; Lin, Li-Zhu; Cai, Chen-Leng; Lu, Min Min; Reth, Michael; Platoshyn, Oleksandr; Yuan, Jason X.-J.; Evans, Sylvia; Chien, Kenneth R.

2017-01-01

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases1–3. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart4. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease. PMID:15703750

Biallelic mutations in IRF8 impair human NK cell maturation and function

PubMed Central

Mace, Emily M.; Gunesch, Justin T.; Chinn, Ivan K.; Angelo, Laura S.; Maisuria, Sheetal; Keller, Michael D.; Togi, Sumihito; Watkin, Levi B.; LaRosa, David F.; Jhangiani, Shalini N.; Muzny, Donna M.; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B.; Hernández-Sanabria, Mayra; Le, Duy T.; Hogg, Graham D.; Cao, Tram N.; Freud, Aharon G.; Szymanski, Eva P.; Collin, Matthew; Cant, Andrew J.; Gibbs, Richard A.; Holland, Steven M.; Caligiuri, Michael A.; Ozato, Keiko; Paust, Silke; Doody, Gina M.; Lupski, James R.; Orange, Jordan S.

2016-01-01

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8–/–, but not Irf8+/–, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense. PMID:27893462

Biallelic mutations in IRF8 impair human NK cell maturation and function.

PubMed

Mace, Emily M; Bigley, Venetia; Gunesch, Justin T; Chinn, Ivan K; Angelo, Laura S; Care, Matthew A; Maisuria, Sheetal; Keller, Michael D; Togi, Sumihito; Watkin, Levi B; LaRosa, David F; Jhangiani, Shalini N; Muzny, Donna M; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B; Hernández-Sanabria, Mayra; Le, Duy T; Hogg, Graham D; Cao, Tram N; Freud, Aharon G; Szymanski, Eva P; Savic, Sinisa; Collin, Matthew; Cant, Andrew J; Gibbs, Richard A; Holland, Steven M; Caligiuri, Michael A; Ozato, Keiko; Paust, Silke; Doody, Gina M; Lupski, James R; Orange, Jordan S

2017-01-03

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.

Recent thymic emigrants and mature naïve T cells exhibit differential DNA methylation at key cytokine loci

PubMed Central

Berkley, Amy M.; Hendricks, Deborah W.; Simmons, Kalynn B.; Fink, Pamela J.

2013-01-01

Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery, and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naïve peripheral T cell pool. We show here that the Il2 and Il4 promoter regions of naïve CD4+ RTEs are characterized by site-specific hypermethylation compared to those of both mature naïve (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared to MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for post-thymic maturation. PMID:23686491

Alterations in dendritic cell function in aged mice: potential implications for immunotherapy design.

PubMed

Paula, Carine; Motta, Adriana; Schmitz, Carla; Nunes, Claudia P; Souza, Ana Paula; Bonorino, Cristina

2009-02-01

It is known that immune system functions decrease with age, and that adaptive immune responses, especially CD4+ T cell function, seem to be the main affected point in immunity with aging. Dendritic cells (DC) are the major antigen presenting cell (APC), and at least part of the defects observed in adaptive immunity of aged individuals could be due to diminished potential of bone marrow to generate new DC, or defects in DC function. In this study, we investigated if the ability of aged bone marrow (BM) to generate new DC in vitro, as well as aged BM-derived DC responses to lypopolysaccharide (LPS). Because DC are important tools in newly developing anti-tumor therapies, we also studied the ability of aged DC to phagocytose and present antigen from necrotic tumor cells. We found that aged BM generated fewer DC in vitro compared to young BM. While LPS-induced DC maturation is reduced in DC of aged mice, a high TNF-alpha production is observed in aged DC even without LPS stimulation. While phagocytosis of tumor cells is not affected by age, and DC derived from aged BM show a higher TNF-alpha production in response to phagocytosis, presentation of tumor antigens was decreased in aged DC. Because class II upregulation in response to phagocytosis was similar between aged and young DC, this could indicate an age associated processing defect in the exogenous pathway. These findings suggest that age of BM used to generate DC does not impair their phagocytic ability or TNF-alpha production, however leads to a decreased yield in mature DC, reduced response to LPS, and diminished antigen processing/presentation potential. Our results are relevant to optimization DC-based vaccine design for aged populations.

Developmental regulation by cytokines of bone marrow-derived dendritic cells and epidermal Langerhans cells.

PubMed

Yamaguchi, Y

1998-01-01

Dendritic cells (DC) are specialized antigen-presenting cells involved in T cell-mediated immune responses. Differentiation and functional maturation of the DC are now known to be regulated by various cytokines, including TGF-beta1. The experiments of this study examined the effect of other cytokines, such as IL-4, IL-10 and IL-6, on the differentiation and maturation of bone marrow (BM)-derived DC (BM-DC) and epidermal Langerhans cells (LC). When IL-6 or IL-10 was added to cultures of BM cells in the presence of GM-CSF, both cytokines, as in the case of TGF-beta1, suppressed the maturation of DC in terms of the expression of adhesion and costimulatory molecules and T cell-stimulating activity. In contrast, IL-4 was not suppressive but rather supportive for the differentiation of DC. However, these suppressive cytokines hardly counteracted the maturation-inducing activity of TNF-alpha when added to cultures of immature DC. In addition, they appeared to block the overmaturation of DC, which is characterized by a loss of MHC class II molecules. Regarding LC maturation in epidermal cell cultures, IL-6 and IL-10 were inhibitory for the expression of CD86 and CD80 in a dose-dependent fashion. Unlike BM-DC, LC maturation was slightly enhanced by TGF-beta1. The protein antigen-presentation by LC to Th1 clone was not affected by IL-6, but slightly reduced by IL-10. These results suggest that each cytokine contributes to regulate the differentiation and maturation of DC at a different developmental stage.

Scarcity of autoreactive human blood IgA+ memory B cells

PubMed Central

Prigent, Julie; Lorin, Valérie; Kök, Ayrin; Hieu, Thierry; Bourgeau, Salomé

2016-01-01

Class‐switched memory B cells are key components of the “reactive” humoral immunity, which ensures a fast and massive secretion of high‐affinity antigen‐specific antibodies upon antigenic challenge. In humans, IgA class‐switched (IgA+) memory B cells and IgA antibodies are abundant in the blood. Although circulating IgA+ memory B cells and their corresponding secreted immunoglobulins likely possess major protective and/or regulatory immune roles, little is known about their specificity and function. Here, we show that IgA+ and IgG+ memory B‐cell antibodies cloned from the same healthy humans share common immunoglobulin gene features. IgA and IgG memory antibodies have comparable lack of reactivity to vaccines, common mucosa‐tropic viruses and commensal bacteria. However, the IgA+ memory B‐cell compartment contains fewer polyreactive clones and importantly, only rare self‐reactive clones compared to IgG+ memory B cells. Self‐reactivity of IgAs is acquired following B‐cell affinity maturation but not antibody class switching. Together, our data suggest the existence of different regulatory mechanisms for removing autoreactive clones from the IgG+ and IgA+ memory B‐cell repertoires, and/or different maturation pathways potentially reflecting the distinct nature and localization of the cognate antigens recognized by individual B‐cell populations. PMID:27469325

Pre-existing Epithelial Diversity in Normal Human Livers: A Tissue-tethered Cytometric Analysis in Portal/Periportal Epithelial Cells

PubMed Central

Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J.

2012-01-01

Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BEC). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the Canals of Hering and/or metaplasia of pre-existing mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high resolution whole slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes pre-existed in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g. scatter plots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically-associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. Results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bi-potential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Conclusion Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable pre-existent hybrid epithelial diversity in normal human liver. This computationally-enabled tissue analysis approach offers much broader potential beyond the results presented here. PMID:23150208

Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells.

PubMed

Noessner, Elfriede; Gastpar, Robert; Milani, Valeria; Brandl, Anna; Hutzler, Peter J S; Kuppner, Maria C; Roos, Miriam; Kremmer, Elisabeth; Asea, Alexzander; Calderwood, Stuart K; Issels, Rolf D

2002-11-15

Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.

Endogenous antigen tunes the responsiveness of naive B cells but not T cells

PubMed Central

Zikherman, Julie; Parameswaran, Ramya; Weiss, Arthur

2012-01-01

In humans up to 75% of newly generated B cells and about 30% of mature B cells exhibit some degree of autoreactivity1. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model BCR transgenic systems have highlighted the critical role played by functional unresponsiveness or ‘anergy’2,3. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B cell tolerance4,5. However, it remains unclear whether the mature diverse B cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which B cell antigen receptor (BCR) signaling rapidly and robustly induces GFP expression under the control of the Nur77 regulatory region, antigen-dependent and – independent BCR signaling events in vivo during B cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure in turn tunes the responsiveness of BCR signaling in B cells at least partly by down-modulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or ‘anergy’ exists in the mature B cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease. PMID:22902503

The touch dome defines an epidermal niche specialized for mechanosensory signaling

PubMed Central

Doucet, Yanne S.; Woo, Seung-Hyun; Ruiz, Marlon E.; Owens, David M.

2013-01-01

Summary In mammalian skin, Merkel cells are mechanoreceptor cells that are required for the perception of gentle touch. Recent evidence indicates that mature Merkel cells descend from the proliferative layer of skin epidermis; however, the stem cell niche for Merkel cell homeostasis has not been reported. Here, we provide the first genetic evidence for maintenance of mature Merkel cells during homeostasis by Krt17+ stem cells located in epidermal touch domes of hairy skin and in the tips of the rete ridges of glabrous skin. Lineage tracing analysis indicated that the entire pool of mature Merkel cells is turned over every 7–8 weeks in adult epidermis and that Krt17+ stem cells also maintain squamous differentiation in the touch dome and in glabrous skin. Finally, selective genetic ablation of Krt17+ touch dome keratinocytes indicates that these cells, and not mature Merkel cells, are primarily responsible for maintaining innervation of the Merkel cell-neurite complex. PMID:23727240

Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer

DTIC Science & Technology

2007-12-01

CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with... surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the DCs...cell surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the

Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogberg, Helena T.; Department of Physiology, Wenner-Gren Institute, Stockholm University; Kinsner-Ovaskainen, Agnieszka

The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA{sub A} receptors), proliferation and differentiation of astrocytes (GFAP and S100{beta}) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed aftermore » exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA{sub A} receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA{sub A} receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100{beta}) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.« less

Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress

PubMed Central

Rissone, Alberto; Weinacht, Katja Gabriele; la Marca, Giancarlo; Bishop, Kevin; Giocaliere, Elisa; Jagadeesh, Jayashree; Felgentreff, Kerstin; Dobbs, Kerry; Al-Herz, Waleed; Jones, Marypat; Chandrasekharappa, Settara; Kirby, Martha; Wincovitch, Stephen; Simon, Karen Lyn; Itan, Yuval; DeVine, Alex; Schlaeger, Thorsten; Schambach, Axel; Sood, Raman

2015-01-01

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD. PMID:26150473

Mesenchymal stem cells derived from bone marrow of diabetic patients portrait unique markers influenced by the diabetic microenvironment.

PubMed

Phadnis, Smruti M; Ghaskadbi, Surendra M; Hardikar, Anandwardhan A; Bhonde, Ramesh R

2009-01-01

Cellular microenvironment is known to play a critical role in the maintenance of human bone marrow-derived mesenchymal stem cells (BM-MSCs). It was uncertain whether BM-MSCs obtained from a 'diabetic milieu' (dBM-MSCs) offer the same regenerative potential as those obtained from healthy (non-diabetic) individuals (hBM-MSCs). To investigate the effect of diabetic microenvironment on human BM-MSCs, we isolated and characterized these cells from diabetic patients (dBM-MSCs). We found that dBM-MSCs expressed mesenchymal markers such as vimentin, smooth muscle actin, nestin, fibronectin, CD29, CD44, CD73, CD90, and CD105. These cells also exhibited multilineage differentiation potential, as evident from the generation of adipocytes, osteocytes, and chondrocytes when exposed to lineage specific differentiation media. Although the cells were similar to hBM-MSCs, 6% (3/54) of dBM-MSCs expressed proinsulin/C-peptide. Emanating from the diabetic microenvironmental milieu, we analyzed whether in vitro reprogramming could afford the maturation of the islet-like clusters (ICAs) derived from dBM-MSCs. Upon mimicking the diabetic hyperglycemic niche and the supplementation of fetal pancreatic extract, to differentiate dBM-MSCs into pancreatic lineage in vitro, we observed rapid differentiation and maturation of dBM-MSCs into islet-like cell aggregates. Thus, our study demonstrated that diabetic hyperglycemic microenvironmental milieu plays a major role in inducing the differentiation of human BM-MSCs in vivo and in vitro.

Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Min Sook; Woo, Min-Yeong; Department of Biomedical Sciences, The Graduate School, Ajou University

2014-10-01

In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with themore » WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.« less

In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

PubMed

Smith, Michelle J; Webber, Beau R; Mohtashami, Mahmood; Stefanski, Heather E; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R

2015-11-01

T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. © 2015 AlphaMed Press.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daud, A.I.; Bumpus, F.M.; Husain, A.

Ovarian angiotensin I (Ang I)-converting enzyme (ACE), estimated by the specific binding of the ACE inhibitor (125I)iodo-MK-351A, is localized on multiple ovarian structures, including follicular granulosa cells, corpora lutea, terminal epithelium, and ovarian blood vessels, but total ovarian ACE does not display a cyclic pattern of variation during the rat estrous cycle. We have previously shown that ACE is localized on the granulosa cell layer of a subpopulation of rat ovarian follicles. Our present study shows that ovarian granulosa cells contain high affinity (binding site affinity (Kd), approximately 90 pM) and low capacity (binding site density (Bmax), approximately 12 fmol/2.5more » X 10(5) cells) (125I)iodo-MK-351A-binding sites and convert (125I)iodo-Ang I to (125I)iodo-Ang II (greater than 85% of this conversion was inhibited by the ACE inhibitor captopril). Throughout the rat estrous cycle, 94-100% of developing follicles and 89-96% of atretic follicles contained high levels of ACE; however, ACE was either not observed or its levels were very low in preovulatory follicles. These findings indicate the presence of high levels of biologically active ACE on the surface of granulosa cells and suggest a potential role for follicular ACE in early stages of follicular maturation and atresia. Although ACE is known to process a variety of peptides found within the ovary, and these peptides may have opposing effects on follicular maturation, we attempted to define the cumulative effect of ACE inhibition on follicular maturation.« less

DNA topoisomerase I and II expression in drug resistantgerm cell tumours

PubMed Central

Berney, D M; Shamash, J; Gaffney, J; Jordan, S; Oliver, R T D

2002-01-01

A small number of testicular germ cell tumours are refractory to current chemotherapy regimens. DNA topoisomerase I is the target for several new drugs and a potential candidate treatment for chemorefractory germ cell tumours. DNA topoisomerase IIα is the target for etoposide, which is currently used regularly in germ cell tumour treatment. The expression of DNA topoisomerase I and IIα were therefore assessed immunohistochemically in a range of testicular tumours, especially those with persistent malignant elements on retroperitoneal lymph node dissection. Pre-chemotherapy orchidectomy specimens were matched with post-chemotherapy retroperitoneal lymph node dissections to examine changes in expression. There was considerable variation in the expression of topoisomerase I in different tumour types. Both yolk sac tumours and teratoma, mature showed universal expression of topoisomerase I, while 38% of seminomas and 30% of embryonal carcinomas were positive. Strong topoisomerase IIα expression was found in embryonal carcinoma. There was a negative correlation between topoisomerase I and IIα expression (P=0.004) and downregulation of topoisomerase IIα after chemotherapy (P=0.02). Topoisomerase I expression appears to increase in those cases with residual teratoma, mature, but is largely unchanged in those cases remaining as embryonal carcinoma. These results suggest that topoisomerase I inhibitors may be useful in chemorefractory germ cell tumours, especially yolk sac tumours and where there are unresectable residual teratoma, mature deposits. British Journal of Cancer (2002) 21, 624–629. doi:10.1038/sj.bjc.6600472 www.bjcancer.com © 2002 Cancer Research UK PMID:12237772

Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

PubMed Central

Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

2011-01-01

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

Enhanced clonal burst size corrects an otherwise defective memory response by CD8+ recent thymic emigrants

PubMed Central

Deets, Katherine A.; Berkley, Amy M.; Bergsbaken, Tessa; Fink, Pamela J.

2016-01-01

The youngest peripheral T cells (recent thymic emigrants or RTEs) are functionally distinct from naïve T cells that have completed post-thymic maturation. We now assess the RTE memory response, and find that RTEs produced less granzyme B than their mature counterparts during infection, but proliferated more and therefore generated equivalent target killing in vivo. After infection, RTE numbers contracted less dramatically than those of mature T cells, but RTEs were delayed in their transition to central memory, displaying impaired expression of CD62L, IL-2, Eomesodermin, and CXCR4, which resulted in impaired bone marrow localization. RTE-derived and mature memory cells expanded equivalently during rechallenge, indicating the robust proliferative capacity of RTEs was maintained independently of central memory phenotype. Thus, the diminished effector function and delayed central memory differentiation of RTE-derived memory cells are counterbalanced by their increased proliferative capacity, driving the efficacy of the RTE response to that of mature T cells. PMID:26873989

Cutting Edge: Enhanced Clonal Burst Size Corrects an Otherwise Defective Memory Response by CD8+ Recent Thymic Emigrants.

PubMed

Deets, Katherine A; Berkley, Amy M; Bergsbaken, Tessa; Fink, Pamela J

2016-03-15

The youngest peripheral T cells (recent thymic emigrants [RTEs]) are functionally distinct from naive T cells that have completed postthymic maturation. We assessed the RTE memory response and found that RTEs produced less granzyme B than their mature counterparts during infection but proliferated more and, therefore, generated equivalent target killing in vivo. Postinfection, RTE numbers contracted less dramatically than those of mature T cells, but RTEs were delayed in their transition to central memory, displaying impaired expression of CD62L, IL-2, Eomesodermin, and CXCR4, which resulted in impaired bone marrow localization. RTE-derived and mature memory cells expanded equivalently during rechallenge, indicating that the robust proliferative capacity of RTEs was maintained independently of central memory phenotype. Thus, the diminished effector function and delayed central memory differentiation of RTE-derived memory cells are counterbalanced by their increased proliferative capacity, driving the efficacy of the RTE response to that of mature T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

Treatment with acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte quality and subsequent embryonic development.

PubMed

Xu, Hui-Yan; Yang, Xiao-Gan; Lu, Sheng-Sheng; Liang, Xing-Wei; Lu, Yang-Qing; Zhang, Ming; Lu, Ke-Huan

2018-06-01

Oocyte quality is one of the important factors in female fertility, in vitro maturation (IVM), and subsequent embryonic development. In the present study, we assessed whether acetyl-l-carnitine (ALC) supplementation during in vitro maturation of buffalo oocytes could improve oocyte quality and subsequent embryonic development. To determine the optimal level of ALC supplementation, we matured cumulus-oocyte complexes in maturation medium supplemented with 0, 2.5, and 5 mM ALC. The oocytes with a polar body were selected for parthenogenetic activation (PA) and in vitro fertilization (IVF). We found that oocytes matured in 2.5 mM ALC had significantly higher PA blastocyst rate (P < 0.05) and blastocyst cell number than those of unsupplemented oocytes (P < 0.05) and a significantly higher IVF blastocyst rate than that of oocytes matured in 5 mM ALC (P < 0.05). In all further experiments, we supplemented the maturation medium with 2.5 mM ALC. We then tested whether ALC supplementation could improve various markers of oocytes and cumulus cells. We compared cell proliferation; concentrations of reactive oxygen species (ROS), intracellular ATP, estradiol, and progesterone; mitochondrial distribution; mitochondrial DNA copy number (mtDNA); and expression levels of four genes encoding oocyte-derived factors (GDF9, BMP15) and steroid hormones (StAR, P450scc) between the supplemented and unsupplemented oocytes and cumulus cells. Cumulus cells matured with ALC supplementation were more prolific than those matured without ALC supplementation (P < 0.05). Oocytes treated with ALC had lower concentrations of intracellular ROS (P < 0.05) and a higher rate of diffuse mitochondrial distributions (P < 0.05) than those of untreated oocytes. Additionally, the mtDNA was higher in the ALC-treated oocytes (P < 0.05) and cumulus cells (P < 0.05) than that in the untreated cells. The ALC-treated maturation medium had a higher postmaturation concentration of estradiol than that of the untreated medium (P < 0.05). Finally, the gene expression levels of P450scc and GDF9 were greater in ALC-treated oocytes and cumulus cells than those in untreated cells (P < 0.05). Therefore, in buffalo, our results suggest that ALC affects mitochondrial function, regulates oocyte-derived paracrine factors, and increases the production of steroid hormones, leading to increased quality of matured oocytes and improved embryonic development in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, M.; Takeishi, Takashi; Geissler, E.N.

1991-07-15

The authors investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF{sup 164} (rrSCF{sup 164}) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of connective tissue-type mast cells (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF{sup 164} induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mastmore » cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC> BMCMC maintained in rrSCF{sup 164} not only proliferated but also matured. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.« less

No amelioration of uromodulin maturation and trafficking defect by sodium 4-phenylbutyrate in vivo: studies in mouse models of uromodulin-associated kidney disease.

PubMed

Kemter, Elisabeth; Sklenak, Stefanie; Rathkolb, Birgit; Hrabě de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard; Wanke, Ruediger

2014-04-11

Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium 4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as a potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IκB kinase α/β, an indirect activator of NF-κB. Furthermore, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52, and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention.

Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

PubMed

Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

2013-04-01

Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage. Copyright © 2012 AlphaMed Press.

Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection.

PubMed

Adnan, Sama; Reeves, R Keith; Gillis, Jacqueline; Wong, Fay E; Yu, Yi; Camp, Jeremy V; Li, Qingsheng; Connole, Michelle; Li, Yuan; Piatak, Michael; Lifson, Jeffrey D; Li, Wenjun; Keele, Brandon F; Kozlowski, Pamela A; Desrosiers, Ronald C; Haase, Ashley T; Johnson, R Paul

2016-12-01

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.

THY-1 Receptor Expression Differentiates Cardiosphere-Derived Cells with Divergent Cardiogenic Differentiation Potential

PubMed Central

Gago-Lopez, Nuria; Awaji, Obinna; Zhang, Yiqiang; Ko, Christopher; Nsair, Ali; Liem, David; Stempien-Otero, April; MacLellan, W. Robb

2014-01-01

Summary Despite over a decade of intense research, the identity and differentiation potential of human adult cardiac progenitor cells (aCPC) remains controversial. Cardiospheres have been proposed as a means to expand aCPCs in vitro, but the identity of the progenitor cell within these 3D structures is unknown. We show that clones derived from cardiospheres could be subdivided based on expression of thymocyte differentiation antigen 1 (THY-1/CD90) into two distinct populations that exhibit divergent cardiac differentiation potential. One population, which is CD90+, expressed markers consistent with a mesenchymal/myofibroblast cell. The second clone type was CD90− and could form mature, functional myocytes with sarcomeres albeit at a very low rate. These two populations of cardiogenic clones displayed distinct cell surface markers and unique transcriptomes. Our study suggests that a rare aCPC exists in cardiospheres along with a mesenchymal/myofibroblast cell, which demonstrates incomplete cardiac myocyte differentiation. PMID:24936447

Genetic address book for retinal cell types.

PubMed

Siegert, Sandra; Scherf, Brigitte Gross; Del Punta, Karina; Didkovsky, Nick; Heintz, Nathaniel; Roska, Botond

2009-09-01

The mammalian brain is assembled from thousands of neuronal cell types that are organized in distinct circuits to perform behaviorally relevant computations. Transgenic mouse lines with selectively marked cell types would facilitate our ability to dissect functional components of complex circuits. We carried out a screen for cell type-specific green fluorescent protein expression in the retina using BAC transgenic mice from the GENSAT project. Among others, we identified mouse lines in which the inhibitory cell types of the night vision and directional selective circuit were selectively labeled. We quantified the stratification patterns to predict potential synaptic connectivity between marked cells of different lines and found that some of the lines enabled targeted recordings and imaging of cell types from developing or mature retinal circuits. Our results suggest the potential use of a stratification-based screening approach for characterizing neuronal circuitry in other layered brain structures, such as the neocortex.

Canonical transient receptor potential channel 2 (TRPC2): old name-new games. Importance in regulating of rat thyroid cell physiology.

PubMed

Törnquist, Kid; Sukumaran, Pramod; Kemppainen, Kati; Löf, Christoffer; Viitanen, Tero

2014-11-01

In addition to the TSH-cyclic AMP signalling pathway, calcium signalling is of crucial importance in thyroid cells. Although the importance of calcium signalling has been thoroughly investigated for several decades, the nature of the calcium channels involved in signalling is unknown. In a recent series of investigations using the well-studied rat thyroid FRTL-5 cell line, we showed that these cells exclusively express the transient receptor potential canonical 2 (TRPC2) channel. Our results suggested that the TRPC2 channel is of significant importance in regulating thyroid cell function. These investigations were the first to show that thyroid cells express a member of the TRPC family of ion channels. In this review, we will describe the importance of the TRPC2 channel in regulating TSH receptor expression, thyroglobulin maturation, intracellular calcium and iodide homeostasis and that the channel also regulates thyroid cell proliferation.

Survival of mature mouse olfactory sensory neurons labeled genetically perinatally.

PubMed

Holl, Anna-Maria

2018-04-01

The main olfactory epithelium (MOE) of an adult mouse harbors a few million mature olfactory sensory neurons (OSNs), which are traditionally defined as mature by their expression of the olfactory marker protein (OMP). Mature OSNs differentiate in situ from stem cells at the base of the MOE. The consensus view is that mature OSNs have a defined lifespan and then undergo programmed cell death, and that the adult MOE maintains homeostasis by generating new mature OSNs from stem cells. But there is also evidence for mature OSNs that are long-lived. Thus far modern genetic tools have not been applied to quantify survival of a population of OSNs that are mature at a given point in time. Here, a genetic strategy was developed to label irreversibly OMP-expressing OSNs in mice. A gene-targeted OMP-CreERT2 strain was generated in which mature OSNs express an enzymatically inactive version of the Cre recombinase. The fusion protein CreERT2 becomes transiently active when exposed to tamoxifen, and in the presence of a Cre reporter in the genome such as tdRFP, CreERT2-expressing cells become irreversibly labeled. A cohort of mice was generated with the same day of birth by in vitro fertilization and embryo transfer, and injected tamoxifen in their mothers at E18.5 of gestation. I counted RFP immunoreactive cells in the MOE and vomeronasal organ of 36 tamoxifen-exposed OMP-CreERT2 × tdRFP mice from 7 age groups: postnatal day (PD)1.5, PD3.5, PD6.5, 3 weeks, 9 weeks, 6 months, and 12 months. Approximately 7.8% of perinatally labeled cells remain at 12 months, confirming that some mature OSNs are indeed long-lived. The survival curve of the population of perinatally labeled MOE cells can be modeled with a mean half-life of 26 days for the population as a whole, excluding the long-lived cells. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

Assessment of Mouse Germinal Vesicle Stage Oocyte Quality by Evaluating the Cumulus Layer, Zona Pellucida, and Perivitelline Space

PubMed Central

Liu, Ying-Lei; Chen, Ying; Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Liang, Cheng-Guang

2014-01-01

To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications. PMID:25144310

Classification and clinical behavior of blastic plasmacytoid dendritic cell neoplasms according to their maturation-associated immunophenotypic profile

PubMed Central

Martín-Martín, Lourdes; López, Antonio; Vidriales, Belén; Caballero, María Dolores; Rodrigues, António Silva; Ferreira, Silvia Inês; Lima, Margarida; Almeida, Sérgio; Valverde, Berta; Martínez, Pilar; Ferrer, Ana; Candeias, Jorge; Ruíz-Cabello, Francisco; Buadesa, Josefa Marco; Sempere, Amparo; Villamor, Neus

2015-01-01

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of leukemia/lymphoma, whose diagnosis can be difficult to achieve due to its clinical and biological heterogeneity, as well as its overlapping features with other hematologic malignancies. In this study we investigated whether the association between the maturational stage of tumor cells and the clinico-biological and prognostic features of the disease, based on the analysis of 46 BPDCN cases classified into three maturation-associated subgroups on immunophenotypic grounds. Our results show that blasts from cases with an immature plasmacytoid dendritic cell (pDC) phenotype exhibit an uncommon CD56− phenotype, coexisting with CD34+ non-pDC tumor cells, typically in the absence of extramedullary (e.g. skin) disease at presentation. Conversely, patients with a more mature blast cell phenotype more frequently displayed skin/extramedullary involvement and spread into secondary lymphoid tissues. Despite the dismal outcome, acute lymphoblastic leukemia-type therapy (with central nervous system prophylaxis) and/or allogeneic stem cell transplantation appeared to be the only effective therapies. Overall, our findings indicate that the maturational profile of pDC blasts in BPDCN is highly heterogeneous and translates into a wide clinical spectrum -from acute leukemia to mature lymphoma-like behavior-, which may also lead to variable diagnosis and treatment. PMID:26056082

Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies

PubMed Central

Zweidler-McKay, Patrick A.; He, Yiping; Xu, Lanwei; Rodriguez, Carlos G.; Karnell, Fredrick G.; Carpenter, Andrea C.; Aster, Jon C.; Allman, David; Pear, Warren S.

2005-01-01

Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)–translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies. PMID:16118316

A distinct plasmablast and naïve B-cell phenotype in primary immune thrombocytopenia

PubMed Central

Flint, Shaun M.; Gibson, Adele; Lucas, Geoff; Nandigam, Raghava; Taylor, Louise; Provan, Drew; Newland, Adrian C.; Savage, Caroline O.; Henderson, Robert B.

2016-01-01

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4+ T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95+ naïve B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naïve B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naïve B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia. PMID:26969086

A distinct plasmablast and naïve B-cell phenotype in primary immune thrombocytopenia.

PubMed

Flint, Shaun M; Gibson, Adele; Lucas, Geoff; Nandigam, Raghava; Taylor, Louise; Provan, Drew; Newland, Adrian C; Savage, Caroline O; Henderson, Robert B

2016-06-01

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4(+) T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95(+) naïve B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naïve B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naïve B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia. Copyright© Ferrata Storti Foundation.

TNF-α and Tumor Lysate Promote the Maturation of Dendritic Cells for Immunotherapy for Advanced Malignant Bone and Soft Tissue Tumors

PubMed Central

Miwa, Shinji; Nishida, Hideji; Tanzawa, Yoshikazu; Takata, Munetomo; Takeuchi, Akihiko; Yamamoto, Norio; Shirai, Toshiharu; Hayashi, Katsuhiro; Kimura, Hiroaki; Igarashi, Kentaro; Mizukoshi, Eishiro; Nakamoto, Yasunari; Kaneko, Shuichi; Tsuchiya, Hiroyuki

2012-01-01

Background Dendritic cells (DCs) play a pivotal role in the immune system. There are many reports concerning DC-based immunotherapy. The differentiation and maturation of DCs is a critical part of DC-based immunotherapy. We investigated the differentiation and maturation of DCs in response to various stimuli. Methods Thirty-one patients with malignant bone and soft tissue tumors were enrolled in this study. All the patients had metastatic tumors and/or recurrent tumors. Peripheral blood mononuclear cells (PBMCs) were suspended in media containing interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF). These cells were then treated with or without 1) tumor lysate (TL), 2) TL + TNF-α, 3) OK-432. The generated DCs were mixed and injected in the inguinal or axillary region. Treatment courses were performed every week and repeated 6 times. A portion of the cells were analyzed by flow cytometry to determine the degree of differentiation and maturation of the DCs. Serum IFN-γ and serum IL-12 were measured in order to determine the immune response following the DC-based immunotherapy. Results Approximately 50% of PBMCs differentiated into DCs. Maturation of the lysate-pulsed DCs was slightly increased. Maturation of the TL/TNF-α-pulsed DCs was increased, commensurate with OK-432-pulsed DCs. Serum IFN-γ and serum IL-12 showed significant elevation at one and three months after DC-based immunotherapy. Conclusions Although TL-pulsed DCs exhibit tumor specific immunity, TL-pulsed cells showed low levels of maturation. Conversely, the TL/TNF-α-pulsed DCs showed remarkable maturation. The combination of IL-4/GM-CSF/TL/TNF-α resulted in the greatest differentiation and maturation for DC-based immunotherapy for patients with bone and soft tissue tumors. PMID:23300824

Regulation of spinogenesis in mature Purkinje cells via mGluR/PKC-mediated phosphorylation of CaMKIIβ

PubMed Central

Sugawara, Takeyuki; Hisatsune, Chihiro; Miyamoto, Hiroyuki; Ogawa, Naoko; Mikoshiba, Katsuhiko

2017-01-01

Dendritic spines of Purkinje cells form excitatory synapses with parallel fiber terminals, which are the primary sites for cerebellar synaptic plasticity. Nevertheless, how density and morphology of these spines are properly maintained in mature Purkinje cells is not well understood. Here we show an activity-dependent mechanism that represses excessive spine development in mature Purkinje cells. We found that CaMKIIβ promotes spine formation and elongation in Purkinje cells through its F-actin bundling activity. Importantly, activation of group I mGluR, but not AMPAR, triggers PKC-mediated phosphorylation of CaMKIIβ, which results in dissociation of the CaMKIIβ/F-actin complex. Defective function of the PKC-mediated CaMKIIβ phosphorylation promotes excess F-actin bundling and leads to abnormally numerous and elongated spines in mature IP3R1-deficient Purkinje cells. Thus, our data suggest that phosphorylation of CaMKIIβ through the mGluR/IP3R1/PKC signaling pathway represses excessive spine formation and elongation in mature Purkinje cells. PMID:28607044

Dietary Restriction and Fasting Arrest B and T Cell Development and Increase Mature B and T Cell Numbers in Bone Marrow

PubMed Central

Shushimita, Shushimita; de Bruijn, Marjolein J. W.; de Bruin, Ron W. F.; IJzermans, Jan N. M.; Hendriks, Rudi W.; Dor, Frank J. M. F.

2014-01-01

Dietary restriction (DR) delays ageing and extends life span. Both long- and short-term DR, as well as short-term fasting provide robust protection against many “neuronal and surgery related damaging phenomena” such as Parkinson’s disease and ischemia-reperfusion injury. The exact mechanism behind this phenomenon has not yet been elucidated. Its anti-inflammatory actions prompted us to thoroughly investigate the consequences of DR and fasting on B and T cell compartments in primary and secondary lymphoid organs of male C57Bl/6 mice. In BM we found that DR and fasting cause a decrease in the total B cell population and arrest early B cell development, while increasing the number of recirculating mature B cells. In the fasting group, a significant reduction in peripheral B cell counts was observed in both spleen and mesenteric lymph nodes (mLN). Thymopoiesis was arrested significantly at double negative DN2 stage due to fasting, whereas DR resulted in a partial arrest of thymocyte development at the DN4 stage. Mature CD3+ T cell populations were increased in BM and decreased in both spleen and mLN. Thus, DR arrests B cell development in the BM but increases the number of recirculating mature B cells. DR also arrests maturation of T cells in thymus, resulting in depletion of mature T cells from spleen and mLN while recruiting them to the BM. The functional relevance in relation to protection against organ damage needs to be determined. PMID:24504160

Microfluidic assay of the deformability of primitive erythroblasts.

PubMed

Zhou, Sitong; Huang, Yu-Shan; Kingsley, Paul D; Cyr, Kathryn H; Palis, James; Wan, Jiandi

2017-09-01

Primitive erythroblasts (precursors of red blood cells) enter vascular circulation during the embryonic period and mature while circulating. As a result, primitive erythroblasts constantly experience significant hemodynamic shear stress. Shear-induced deformation of primitive erythroblasts however, is poorly studied. In this work, we examined the deformability of primitive erythroblasts at physiologically relevant flow conditions in microfluidic channels and identified the regulatory roles of the maturation stage of primitive erythroblasts and cytoskeletal protein 4.1 R in shear-induced cell deformation. The results showed that the maturation stage affected the deformability of primitive erythroblasts significantly and that primitive erythroblasts at later maturational stages exhibited a better deformability due to a matured cytoskeletal structure in the cell membrane.

A mucin-like peptide from Fasciola hepatica instructs dendritic cells with parasite specific Th1-polarizing activity.

PubMed

Noya, Verónica; Brossard, Natalie; Rodríguez, Ernesto; Dergan-Dylon, L Sebastián; Carmona, Carlos; Rabinovich, Gabriel A; Freire, Teresa

2017-01-12

Fasciolosis is a trematode zoonosis of interest in public health and cattle production. We report here the immunostimulatory effect of a 66 mer mucin-like peptide from Fasciola hepatica (Fhmuc), which synergizes with lipopolysaccharide (LPS) to promote dendritic cell (DC) maturation, endowing these cells with Th1-polarizing capacity. Exposure of DCs to Fhmuc in presence of LPS induced enhanced secretion of pro-inflammatory cytokines and expression of co-stimulatory molecules by DCs, promoting their T cell stimulatory capacity and selectively augmenting IFN-γ secretion by allogeneic T cells. Furthermore, exposure of DCs to Fhmuc augmented LPS-induced Toll-like receptor (TLR) 4 expression on the cell surface. Finally, Fhmuc-conditioned DCs induced parasite specific-adaptive immunity with increased levels of IFN-γ secreted by splenocytes from vaccinated animals, and higher parasite-specific IgG antibodies. However, Fhmuc-treated DC conferred modest protection against F. hepatica infection highlighting the potent immuno-regulatory capacity of the parasite. In summary, this work highlights the capacity of a mucin-derived peptide from F. hepatica to enhance LPS-maturation of DCs and induce parasite-specific immune responses with potential implications in vaccination and therapeutic strategies.

A mucin-like peptide from Fasciola hepatica instructs dendritic cells with parasite specific Th1-polarizing activity

PubMed Central

Noya, Verónica; Brossard, Natalie; Rodríguez, Ernesto; Dergan-Dylon, L. Sebastián; Carmona, Carlos; Rabinovich, Gabriel A.; Freire, Teresa

2017-01-01

Fasciolosis is a trematode zoonosis of interest in public health and cattle production. We report here the immunostimulatory effect of a 66 mer mucin-like peptide from Fasciola hepatica (Fhmuc), which synergizes with lipopolysaccharide (LPS) to promote dendritic cell (DC) maturation, endowing these cells with Th1-polarizing capacity. Exposure of DCs to Fhmuc in presence of LPS induced enhanced secretion of pro-inflammatory cytokines and expression of co-stimulatory molecules by DCs, promoting their T cell stimulatory capacity and selectively augmenting IFN-γ secretion by allogeneic T cells. Furthermore, exposure of DCs to Fhmuc augmented LPS-induced Toll-like receptor (TLR) 4 expression on the cell surface. Finally, Fhmuc-conditioned DCs induced parasite specific-adaptive immunity with increased levels of IFN-γ secreted by splenocytes from vaccinated animals, and higher parasite-specific IgG antibodies. However, Fhmuc-treated DC conferred modest protection against F. hepatica infection highlighting the potent immuno-regulatory capacity of the parasite. In summary, this work highlights the capacity of a mucin-derived peptide from F. hepatica to enhance LPS-maturation of DCs and induce parasite-specific immune responses with potential implications in vaccination and therapeutic strategies. PMID:28079156

Mitoquinone restores platelet production in irradiation-induced thrombocytopenia

PubMed Central

Ramsey, Haley; Zhang, Qi; Wu, Mei X.

2014-01-01

Myelodysplastic syndromes (MDS) are hallmarked by cytopenia and dysplasia of hematopoietic cells, often accompanied by mitochondrial dysfunction and increases of reactive oxygen species (ROS) within affected cells. However, it is not known whether the increase in ROS production is an instigator or a byproduct of the disease. The present investigation shows that mice lacking immediate early responsive gene X-1 (IEX-1) exhibit lineage specific increases in ROS production and abnormal cytology upon radiation in blood cell types commonly identified in MDS. These affected cell lineages chiefly have the bone marrow as a primary site of differentiation and maturation, while cells with extramedullary differentiation and maturation like B- and T-cells remain unaffected. Increased ROS production is likely to contribute significantly to irradiation-induced thrombocytopenia in the absence of IEX-1 as demonstrated by effective reversal of the disorder after mitoquinone (MitoQ) treatment, a mitochondria-specific antioxidant. MitoQ reduced intracellular ROS production within megakaryocytes and platelets. It also normalized mitochondrial membrane potential and superoxide production in platelets in irradiated, IEX-1 deficient mice. The lineage-specific effects of mitochondrial ROS may help us understand the etiology of thrombocytopenia in association with MDS in a subgroup of the patients. PMID:25025394

Rag Deletion in Peripheral T Cells Blocks TCR Revision

PubMed Central

Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

2010-01-01

Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

Transcription Factor Foxo1 Is a Negative Regulator of NK Cell Maturation and Function

PubMed Central

Deng, Youcai; Kerdiles, Yann; Chu, Jianhong; Yuan, Shunzong; Wang, Youwei; Chen, Xilin; Mao, Hsiaoyin; Zhang, Lingling; Zhang, Jianying; Hughes, Tiffany; Deng, Yafei; Zhang, Qi; Wang, Fangjie; Zou, Xianghong; Liu, Chang-Gong; Freud, Aharon G.; Li, Xiaohui; Caligiuri, Michael A; Vivier, Eric; Yu, Jianhua

2015-01-01

SUMMARY Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes through upregulating CD62L expression, and impaired late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21+/− mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions. PMID:25769609

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung

PubMed Central

Lange, Alexander W.; Sridharan, Anusha; Xu, Yan; Stripp, Barry R.; Perl, Anne-Karina; Whitsett, Jeffrey A.

2015-01-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. PMID:25480985

Enhanced elastin synthesis and maturation in human vascular smooth muscle tissue derived from induced-pluripotent stem cells.

PubMed

Eoh, Joon H; Shen, Nian; Burke, Jacqueline A; Hinderer, Svenja; Xia, Zhiyong; Schenke-Layland, Katja; Gerecht, Sharon

2017-04-01

Obtaining vascular smooth muscle tissue with mature, functional elastic fibers is a key obstacle in tissue-engineered blood vessels. Poor elastin secretion and organization leads to a loss of specialization in contractile smooth muscle cells, resulting in over proliferation and graft failure. In this study, human induced-pluripotent stem cells (hiPSCs) were differentiated into early smooth muscle cells, seeded onto a hybrid poly(ethylene glycol) dimethacrylate/poly (l-lactide) (PEGdma-PLA) scaffold and cultured in a bioreactor while exposed to pulsatile flow, towards maturation into contractile smooth muscle tissue. We evaluated the effects of pulsatile flow on cellular organization as well as elastin expression and assembly in the engineered tissue compared to a static control through immunohistochemistry, gene expression and functionality assays. We show that culturing under pulsatile flow resulted in organized and functional hiPSC derived smooth muscle tissue. Immunohistochemistry analysis revealed hiPSC-smooth muscle tissue with robust, well-organized cells and elastic fibers and the supporting microfibril proteins necessary for elastic fiber assembly. Through qRT-PCR analysis, we found significantly increased expression of elastin, fibronectin, and collagen I, indicating the synthesis of necessary extracellular matrix components. Functionality assays revealed that hiPSC-smooth muscle tissue cultured in the bioreactor had an increased calcium signaling and contraction in response to a cholinergic agonist, significantly higher mature elastin content and improved mechanical properties in comparison to the static control. The findings presented here detail an effective approach to engineering elastic human vascular smooth muscle tissue with the functionality necessary for tissue engineering and regenerative medicine applications. Obtaining robust, mature elastic fibers is a key obstacle in tissue-engineered blood vessels. Human induced-pluripotent stem cells have become of interest due to their ability to supplement tissue engineered scaffolds. Their ability to differentiate into cells of vascular lineages with defined phenotypes serves as a potential solution to a major cause of graft failure in which phenotypic shifts in smooth muscle cells lead to over proliferation and occlusion of the graft. Herein, we have differentiated human induced-pluripotent stem cells in a pulsatile flow bioreactor, resulting in vascular smooth muscle tissue with robust elastic fibers and enhanced functionality. This study highlights an effective approach to engineering elastic functional vascular smooth muscle tissue for tissue engineering and regenerative medicine applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Targeting Toll-like receptor 7/8 enhances uptake of apoptotic leukemic cells by monocyte-derived dendritic cells but interferes with subsequent cytokine-induced maturation.

PubMed

van den Ancker, Willemijn; van Luijn, Marvin M; Ruben, Jurjen M; Westers, Theresia M; Bontkes, Hetty J; Ossenkoppele, Gert J; de Gruijl, Tanja D; van de Loosdrecht, Arjan A

2011-01-01

Therapeutic vaccination with dendritic cells (DC) is an emerging investigational therapy for eradication of minimal residual disease in acute myeloid leukemia. Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with leukemia-associated antigens (LAA). However, the optimal source of LAA and the choice of DC-activating stimuli are still not well defined. Here, loading with leukemic cell preparations (harboring both unknown and known LAA) was explored in combination with a DC maturation-inducing cytokine cocktail (CC; IL-1β, IL-6, TNF-α, and PGE(2)) and Toll-like receptor ligands (TLR-L) to optimize uptake. Since heat shock induced apoptotic blasts were more efficiently taken up than lysates, we focused on uptake of apoptotic leukemic cells. Uptake of apoptotic blast was further enhanced by the TLR7/8-L R848 (20-30%); in contrast, CC-induced maturation inhibited uptake. CC, and to a lesser extent R848, enhanced the ability of MoDC to migrate and stimulate T cells. Furthermore, class II-associated invariant chain peptide expression was down-modulated after R848- or CC-induced maturation, indicating enhanced processing and presentation of antigenic peptides. To improve both uptake and maturation, leukemic cells and MoDC were co-incubated with R848 for 24 h followed by addition of CC. However, this approach interfered with CC-mediated MoDC maturation as indicated by diminished migratory and T cell stimulatory capacity, and the absence of IL-12 production. Taken together, our data demonstrate that even though R848 improved uptake of apoptotic leukemic cells, the sequential use of R848 and CC is counter-indicated due to its adverse effects on MoDC maturation.

Concurrent primary carcinoid tumor arising within mature teratoma and clear cell renal cell carcinoma in the horseshoe kidney: report of a rare case and review of the literature.

PubMed

Sun, Ke; You, Qihan; Zhao, Ming; Yao, Hongtian; Xiang, Hua; Wang, Lijun

2013-01-01

Primary carcinoid tumor arising in a mature teratoma of the horseshoe kidney is exceptionally rare and only 4 such cases have been reported in the world literature to date. The simultaneous occurrence of different subtypes of renal cell carcinoma (RCC) or RCC coexistence with non-RCC neoplasms from the same kidney is unusual and infrequently reported. Herein we report a case of primary carcinoid tumor arising within mature teratoma, concurrent with a clear cell RCC in the horseshoe kidney of a 37-year-old man. Histologically, both the carcinoid tumor and clear cell RCC demonstrated the characteristic morphology in their classic forms. In addition to the carcinoid tumor, the mature teratoma consisted of variably sized, large cystic spaces lined by cytologically bland mucinous columnar epithelium, pseudostratified columnar epithelium, ciliated epithelium and mature smooth muscle fibers were also identified within the cystic wall. Furthermore, foci of round, small nodules composed of mature prostatic acinus were noted in the teratoma which was confirmed by exhibiting strong immunoreactivity for prostate specific antigen. The present case serves to expand the histologic component that may be encountered in the mature terotoma of the kidney and further broadens the spectrum of primary tumors occurring in the horseshoe kidney.

Postnatal development, maturation and aging in the mouse cochlea and their effects on hair cell regeneration

PubMed Central

Walters, Brad; Zuo, Jian

2012-01-01

The organ of Corti in the mammalian inner ear is comprised of mechanosensory hair cells (HCs) and nonsensory supporting cells (SCs), both of which are believed to be terminally postmitotic beyond late embryonic ages. Consequently, regeneration of HCs and SCs does not occur naturally in the adult mammalian cochlea, though recent evidence suggests that these cells may not be completely or irreversibly quiescent in at earlier postnatal ages. Furthermore, regenerative processes can be induced by genetic and pharmacological manipulations, but, more and more reports suggest that regenerative potential declines as the organ of Corti continues to age. In numerous mammalian systems, such effects of aging on regenerative potential are well established. However, in the cochlea, the problem of regeneration has not been traditionally viewed as one of aging. This is an important consideration as current models are unable to elicit widespread regeneration or full recovery of function at adult ages yet regenerative therapies will need to be developed specifically for adult populations. Still, the advent of gene targeting and other genetic manipulations has established mice as critically important models for the study of cochlear development and HC regeneration and suggests that auditory HC regeneration in adult mammals may indeed be possible. Thus, this review will focus on the pursuit of regeneration in the postnatal and adult mouse cochlea and highlight processes that occur during postnatal development, maturation, and aging that could contribute to an age-related decline in regenerative potential. Second, we will draw upon the wealth of knowledge pertaining to age related senescence in tissues outside of the ear to synthesize new insights and potentially guide future research aimed at promoting HC regeneration in the adult cochlea. PMID:23164734

Effect of delignification upon in vitro digestion of forage cellulose.

PubMed

Darcy, B K; Belyea, R L

1980-10-01

Orchardgrass forages harvested at two maturities (early and late) were ground through two screens (1 and 8 mm) and digested in vitro as intact forage and forage delignified by permanganate oxidation. Initial and residual cell wall, initial and residual cellulose and potentially digestible cellulose were greater in late intact forage than in the early. In the delignified forage, late cut forage had less residual cellulose than did the early, but initial and potentially digestible cellulose were similar. Particle size had less consistent and smaller effects upon cell wall and cellulose than did maturity. Cellulose of intact orchardgrass was 64% digested at 72 h vs 94% for cellulose of delignified orchardgrass. Digestion rate of cellulose was .0197 and .0220 logn units/hr for early and late cut intact forage and .0554 and .0719 logn units/hr for early and late cut delignified forage. Removal of the inhibitory effects of lignin increased the amount of digestible cellulose, increased the rate at which cellulose degraded and decreased the indigestible cellulose residue. Reduction in lignin could greatly improve forage intake and utilization at moderate levels of animal production.

Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.

PubMed

Bonakdar, Shahin; Mahmoudi, Morteza; Montazeri, Leila; Taghipoor, Mojtaba; Bertsch, Arnaud; Shokrgozar, Mohammad Ali; Sharifi, Shahriar; Majidi, Mohammad; Mashinchian, Omid; Hamrang Sekachaei, Mohammad; Zolfaghari, Pegah; Renaud, Philippe

2016-06-08

Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

PubMed

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo; Loncarek, Jadranka

2014-09-29

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles

PubMed Central

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo

2014-01-01

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells. PMID:25246616

Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

NASA Astrophysics Data System (ADS)

Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

2016-07-01

A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

Simulation of B Cell Affinity Maturation Explains Enhanced Antibody Cross-Reactivity Induced by the Polyvalent Malaria Vaccine AMA1

DTIC Science & Technology

2014-07-01

cell decisions in lymphoid tissue. Mol. Cell . Biol. 28: 4040–4051. 22. Kosmrlj, A., A. K. Jha, E. S. Huseby, M. Kardar, and A. K. Chakraborty. 2008. How...JUL 2014 2. REPORT TYPE 3. DATES COVERED 00-00-2014 to 00-00-2014 4. TITLE AND SUBTITLE Simulation of B Cell Affinity Maturation Explains...8-98) Prescribed by ANSI Std Z39-18 The Journal of Immunology Simulation of B Cell Affinity Maturation Explains Enhanced Antibody Cross-Reactivity

AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

PubMed Central

Behnke, O.; Moe, H.

1964-01-01

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells. PMID:14206428

Proteomic changes during intestinal cell maturation in vivo

PubMed Central

Chang, Jinsook; Chance, Mark R.; Nicholas, Courtney; Ahmed, Naseem; Guilmeau, Sandra; Flandez, Marta; Wang, Donghai; Byun, Do-Sun; Nasser, Shannon; Albanese, Joseph M.; Corner, Georgia A.; Heerdt, Barbara G.; Wilson, Andrew J.; Augenlicht, Leonard H.; Mariadason, John M.

2008-01-01

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated. PMID:18824147

Chronic corticosterone administration reduces dendritic complexity in mature, but not young granule cells in the rat dentate gyrus.

PubMed

Yau, Suk-Yu; Li, Ang; Tong, Jian-Bin; Bostrom, Crystal; Christie, Brian R; Lee, Tatia M C; So, Kwok-Fai

2016-09-21

Our previous work has shown that exposure to the stress hormone corticosterone (40 mg/kg CORT) for two weeks induces dendritic atrophy of pyramidal neurons in the hippocampal CA3 region and behavioral deficits. However, it is unclear whether this treatment also affects the dentate gyrus (DG), a subregion of the hippocampus comprising a heterogeneous population of young and mature neurons. We examined the effect of CORT treatment on the dendritic complexity of mature and young granule cells in the DG. We utilized a Golgi staining method to investigate the dendritic morphology and spine density of young neurons in the inner granular cell layer (GCL) and mature neurons in the outer GCL in response to CORT application. The expressions of glucocorticoid receptors during neuronal maturation were examined using Western blot analysis in a primary hippocampal neuronal culture. Sholl analysis revealed that CORT treatment decreased the number of intersections and shortened the dendritic length in mature, but not young, granule cells. However, the spine density of mature and young neurons was not affected. Western blot analysis showed a progressive increase in the protein levels of glucocorticoid receptors (GRs) in the cultured primary hippocampal neurons during neuronal maturation. These data suggest that mature neurons are likely more vulnerable to chronic exposure to CORT; this may be due to their higher expression of GRs when compared to younger DG neurons.

Depolarization Alters Phenotype, Maintains Plasticity of Predifferentiated Mesenchymal Stem Cells

PubMed Central

Sundelacruz, Sarah; Levin, Michael

2013-01-01

Although adult stem cell transplantation has been implemented as a therapy for tissue repair, it is limited by the availability of functional adult stem cells. A potential approach to generate stem and progenitor cells may be to modulate the differentiated status of somatic cells. Therefore, there is a need for a better understanding of how the differentiated phenotype of mature cells is regulated. We hypothesize that bioelectric signaling plays an important role in the maintenance of the differentiated state, as it is a functional regulator of the differentiation process in various cells and tissues. In this study, we asked whether the mature phenotype of osteoblasts and adipocytes derived from human mesenchymal stem cells (hMSCs) could be altered by modulation of their membrane potential. hMSC-derived osteoblasts and adipocytes were depolarized by treatment with ouabain, a Na+/K+ ATPase inhibitor, or by treatment with high concentrations of extracellular K+. To characterize the effect of voltage modulation on the differentiated state, the depolarized cells were evaluated for (1) the loss of differentiation markers; (2) the up-regulation of stemness markers and stem properties; and (3) differences in gene expression profiles in response to voltage modulation. hMSC-derived osteoblasts and adipocytes exhibited significant down-regulation of bone and fat tissue markers in response to depolarization, despite the presence of differentiation-inducing soluble factors, suggesting that bioelectric signaling overrides biochemical signaling in the maintenance of cell state. Suppression of the osteoblast or adipocyte phenotype was not accompanied by up-regulation of genes associated with the stem state. Thus, depolarization does not activate the stem cell genetic signature and, therefore, does not induce a full reprogramming event. However, after transdifferentiating the depolarized cells to evaluate for multi-lineage potential, depolarized osteoblasts demonstrated improved ability to achieve correct adipocyte morphology compared with nondepolarized osteoblasts. The present study thus demonstrates that depolarization reduces the differentiated phenotype of hMSC-derived cells and improves their transdifferentiation capacity, but does not restore a stem-like genetic profile. Through global transcript profiling of depolarized osteoblasts, we identified pathways that may mediate the effects of voltage signaling on cell state, which will require a detailed mechanistic inquiry in future studies. PMID:23738690

HIERARCHICAL ORGANIZATION OF OSTEOBLASTS REVEALS THE SIGNIFICANT ROLE OF CD166 IN HEMATOPOIETIC STEM CELL MAINTANANCE AND FUNCTION

PubMed Central

Chitteti, Brahmananda R.; Cheng, Ying-Hua; Kacena, Melissa A.; Srour, Edward F.

2013-01-01

The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166 expression on OB is directly correlated with Runx2 expression and high hematopoiesis enhancing activity (HEA). Fractionation of OB with lineage markers: Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166− cells (CD166−) expressed high OC levels suggesting that CD166− OB are more mature. In vitro hematopoietic potential of LSK cells co-cultured for 7 days with fresh OB or OB pre-cultured for 1, 2, or 3 weeks declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Importantly, LSK cells co-cultured with CD166+CD44+CD90+ OB maintained their in vivo repopulating potential through primary and secondary transplantation, suggesting that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. These studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined definition of OB that can mediate HEA. PMID:23369988

Placing Ion Channels into a Signaling Network of T Cells: From Maturing Thymocytes to Healthy T Lymphocytes or Leukemic T Lymphoblasts

PubMed Central

Delgado-Enciso, Iván; Best-Aguilera, Carlos; Rojas-Sotelo, Rocío Monserrat; Pottosin, Igor

2015-01-01

T leukemogenesis is a multistep process, where the genetic errors during T cell maturation cause the healthy progenitor to convert into the leukemic precursor that lost its ability to differentiate but possesses high potential for proliferation, self-renewal, and migration. A new misdirecting “leukemogenic” signaling network appears, composed by three types of participants which are encoded by (1) genes implicated in determined stages of T cell development but deregulated by translocations or mutations, (2) genes which normally do not participate in T cell development but are upregulated, and (3) nondifferentially expressed genes which become highly interconnected with genes expressed differentially. It appears that each of three groups may contain genes coding ion channels. In T cells, ion channels are implicated in regulation of cell cycle progression, differentiation, activation, migration, and cell death. In the present review we are going to reveal a relationship between different genetic defects, which drive the T cell neoplasias, with calcium signaling and ion channels. We suggest that changes in regulation of various ion channels in different types of the T leukemias may provide the intracellular ion microenvironment favorable to maintain self-renewal capacity, arrest differentiation, induce proliferation, and enhance motility. PMID:25866806

Cutting edge: double-stranded DNA breaks in the IgV region gene were detected at lower frequency in affinity-maturation impeded GANP-/- mice.

PubMed

Kawatani, Yousuke; Igarashi, Hideya; Matsui, Takeshi; Kuwahara, Kazuhiko; Fujimura, Satoru; Okamoto, Nobukazu; Takagi, Katsumasa; Sakaguchi, Nobuo

2005-11-01

Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken gamma-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP-/- B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.

Cutting Edge: Rag deletion in peripheral T cells blocks TCR revision.

PubMed

Hale, J Scott; Ames, Kristina T; Boursalian, Tamar E; Fink, Pamela J

2010-06-01

Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice, this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells.

Neurotrophin Propeptides: Biological Functions and Molecular Mechanisms.

PubMed

Rafieva, Lola M; Gasanov, Eugene V

2016-01-01

Neurotrophins constitute a family of growth factors that play a key role in the regulation of the development and function of the central and peripheral nervous systems. A common feature of all the neurotrophins is their synthesis in cells as long precursors (pre-pro-neurotrophins) that contain an N-terminal signal peptide, a following propeptide and the mature neurotrophin. Although the signal peptide functions have been well studied, the role of neurotrophin propeptides is not so clear. Here, we briefly summarize the biochemistry of neurotrophin propeptides, including their role as folding-assistants for the mature factor and their role in processing and in secretion of neurotrophins. In the main part of the review we summarize our current state of knowledge of the biological activity of neurotrophin propeptides, their possible mechanisms of action, and their potential influence on the activity of the mature neurotrophins.

Notch3 is required for arterial identity and maturation of vascular smooth muscle cells

PubMed Central

Domenga, Valérie; Fardoux, Peggy; Lacombe, Pierre; Monet, Marie; Maciazek, Jacqueline; Krebs, Luke T.; Klonjkowski, Bernard; Berrou, Eliane; Mericskay, Matthias; Li, Zhen; Tournier-Lasserve, Elisabeth; Gridley, Thomas; Joutel, Anne

2004-01-01

Formation of a fully functional artery proceeds through a multistep process. Here we show that Notch3 is required to generate functional arteries in mice by regulating arterial differentiation and maturation of vascular smooth muscle cells (vSMC). In adult Notch3–/– mice distal arteries exhibit structural defects and arterial myogenic responses are defective. The postnatal maturation stage of vSMC is deficient in Notch3–/– mice. We further show that Notch3 is required for arterial specification of vSMC but not of endothelial cells. Our data reveal Notch3 to be the first cell-autonomous regulator of arterial differentiation and maturation of vSMC. PMID:15545631

Determination of the Role of Estrogen Receptors and Estrogen Regulated Genes in B Cell Autoreactivity

DTIC Science & Technology

2011-07-01

Betty Diamond – DOD FINAL REPORT 9 Figure 3: (A) expression of estrogen receptors ERalpha( Esr1 ) and ERbeta (Esr2) in splenic B cells and (B...Urinary 16 OH-Estradiol metabolite in BALB/c and C57BL6 mice. Esr1 0 0.05 0.1 0.15 0.2 Transit. Mature Transit. Mature Transit. Mature Transit. mature P E2

Investigation for the differentiation process of mouse ES cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Yamaguchi, Yoshinori; El-Hagrasy, Maha A.; Shimizu, Eiichi; Saito, Masato; Tamiya, Eiichi

2012-03-01

The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

Location and Dynamics of the Immunodominant CD8 T Cell Response to SIVΔnef Immunization and SIVmac251 Vaginal Challenge

PubMed Central

Sasikala-Appukuttan, Arun K.; Kim, Hyeon O.; Kinzel, Nikilyn J.; Hong, Jung Joo; Smith, Anthony J.; Wagstaff, Reece; Reilly, Cavan; Piatak, Michael; Lifson, Jeffrey D.; Reeves, R. Keith; Johnson, R. Paul; Haase, Ashley T.; Skinner, Pamela J.

2013-01-01

Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission. PMID:24349100

Early embryonic sensitivity to cyclophosphamide in cardiac differentiation from human embryonic stem cells.

PubMed

Zhu, Ming-Xia; Zhao, Jin-Yuan; Chen, Gui-An; Guan, Li

2011-09-01

hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.

Cell Death, Neuronal Plasticity and Functional Loading in the Development of the Central Nervous System

NASA Technical Reports Server (NTRS)

Keefe, J. R.

1985-01-01

Research on the precise timing and regulation of neuron production and maturation in the vestibular and visual systems of Wistar rats and several inbred strains of mice (C57B16 and Pallid mutant) concentrated upon establishing a timing baseline for mitotic development of the neurons of the vestibular nuclei and the peripheral vestibular sensory structures (maculae, cristae). This involved studies of the timing and site of neuronal cell birth and preliminary studies of neuronal cell death in both central and peripheral elements of the mammalian vestibular system. Studies on neuronal generation and maturation in the retina were recently added to provide a mechanism for more properly defining the in utero' developmental age of the individual fetal subject and to closely monitor potential transplacental effects of environmentally stressed maternal systems. Information is given on current efforts concentrating upon the (1) perinatal period of development (E18 thru P14) and (2) the role of cell death in response to variation in the functional loading of the vestibular and proprioreceptive systems in developing mammalian organisms.

Molecular control of steady-state dendritic cell maturation and immune homeostasis.

PubMed

Hammer, Gianna Elena; Ma, Averil

2013-01-01

Dendritic cells (DCs) are specialized sentinels responsible for coordinating adaptive immunity. This function is dependent upon coupled sensitivity to environmental signs of inflammation and infection to cellular maturation-the programmed alteration of DC phenotype and function to enhance immune cell activation. Although DCs are thus well equipped to respond to pathogens, maturation triggers are not unique to infection. Given that immune cells are exquisitely sensitive to the biological functions of DCs, we now appreciate that multiple layers of suppression are required to restrict the environmental sensitivity, cellular maturation, and even life span of DCs to prevent aberrant immune activation during the steady state. At the same time, steady-state DCs are not quiescent but rather perform key functions that support homeostasis of numerous cell types. Here we review these functions and molecular mechanisms of suppression that control steady-state DC maturation. Corruption of these steady-state operatives has diverse immunological consequences and pinpoints DCs as potent drivers of autoimmune and inflammatory disease.

Cutting edge: Contact with secondary lymphoid organs drives postthymic T cell maturation.

PubMed

Houston, Evan G; Nechanitzky, Robert; Fink, Pamela J

2008-10-15

T cell development, originally thought to be completed in the thymus, has recently been shown to continue for several weeks in the lymphoid periphery. The forces that drive this peripheral maturation are unclear. The use of mice transgenic for GFP driven by the RAG2 promoter has enabled the ready identification and analysis of recent thymic emigrants. Here, we show that recent thymic emigrant maturation is a progressive process and is promoted by T cell exit from the thymus. Further, we show that this maturation occurs within secondary lymphoid organs and does not require extensive lymphocyte recirculation.

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina

PubMed Central

Irie, Shoichi; Sanuki, Rikako; Muranishi, Yuki; Kato, Kimiko; Chaya, Taro

2015-01-01

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina. PMID:25986607

Porous hydroxyapatite and biphasic calcium phosphate ceramics promote ectopic osteoblast differentiation from mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Zhang, Lingli; Hanagata, Nobutaka; Maeda, Megumi; Minowa, Takashi; Ikoma, Toshiyuki; Fan, Hongsong; Zhang, Xingdong

2009-04-01

Because calcium phosphate (Ca-P) ceramics have been used as bone substitutes, it is necessary to investigate what effects the ceramics have on osteoblast maturation. We prepared three types of Ca-P ceramics with different Ca-P ratios, i.e. hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), and biphasic calcium phosphate (BCP) ceramics with dense-smooth and porous structures. Comprehensive gene expression microarray analysis of mouse osteoblast-like cells cultured on these ceramics revealed that porous Ca-P ceramics considerably affected the gene expression profiles, having a higher potential for osteoblast maturation. In the in vivo study that followed, porous Ca-P ceramics were implanted into rat skeletal muscle. Sixteen weeks after the implantation, more alkaline-phosphatase-positive cells were observed in the pores of hydroxyapatite and BCP, and the expression of the osteocalcin gene (an osteoblast-specific marker) in tissue grown in pores was also higher in hydroxyapatite and BCP than in β-TCP. In the pores of any Ca-P ceramics, 16 weeks after the implantation, we detected the expressions of marker genes of the early differentiation stage of chondrocytes and the complete differentiation stage of adipocytes, which originate from mesenchymal stem cells, as well as osteoblasts. These marker gene expressions were not observed in the muscle tissue surrounding the implanted Ca-P ceramics. These observations indicate that porous hydroxyapatite and BCP had a greater potential for promoting the differentiation of mesenchymal stem cells into osteoblasts than β-TCP.

PD-1HIGH Follicular CD4 T Helper Cell Subsets Residing in Lymph Node Germinal Centers Correlate with B Cell Maturation and IgG Production in Rhesus Macaques

PubMed Central

Xu, Huanbin; Wang, Xiaolei; Lackner, Andrew A.; Veazey, Ronald S.

2014-01-01

CD4+ T follicular helper (TFH) cells guide development and maturation of B cells and are crucial for effective antibody responses. Here we found rhesus macaque TFH cells, defined as CXCR5+CD4 T cells, contain two major populations: PD-1INT and PD-1HIGH cells. Of these, PD-1HIGHCD4+ T cells highly co-express ICOS but little CCR7, and reside in lymph node germinal centers (GCs), but not in blood. These cells secrete IL-21 and express transcriptional factor Bcl-6 at higher levels than CXCR5+PD-1INTCD4+ T cells. In addition, the frequency of PD-1HIGHCD4+ T cells is low in lymph nodes of newborns, but increases with age. Levels of PD-1HIGHCD4+ T cells correlate with mature B cells in lymph nodes, and PD-1 blockade in PD-1HIGHCD4+ T and B cell co-cultures significantly inhibits IgG production. In summary, PD-1HIGHCD4+ T cells residing in GC represent a specific TFH subset that contributes to maturation of B cells and IgG production. PMID:24678309

Stem cells and reproduction.

PubMed

Du, Hongling; Taylor, Hugh S

2010-06-01

To review the latest developments in reproductive tract stem cell biology. In 2004, two studies indicated that ovaries contain stem cells which form oocytes in adults and that can be cultured in vitro into mature oocytes. A live birth after orthotopic transplantation of cryopreserved ovarian tissue in a woman whose ovaries were damaged by chemotherapy demonstrates the clinical potential of these cells. In the same year, another study provided novel evidence of endometrial regeneration by stem cells in women who received bone marrow transplants. This finding has potential for the use in treatment of uterine disorders. It also supports a new theory for the cause of endometriosis, which may have its origin in ectopic transdifferentiation of stem cells. Several recent studies have demonstrated that fetal cells enter the maternal circulation and generate microchimerism in the mother. The uterus is a dynamic organ permeable to fetal stem cells, capable of transdifferentiation and an end organ in which bone marrow stem cells may differentiate. Finally stem cell transformation can be an underlying cause of ovarian cancer. Whereas we are just beginning to understand stem cells, the potential implications of stem cells to reproductive biology and medicine are apparent.

Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair.

PubMed

Hartman, Matthew E; Dai, Dao-Fu; Laflamme, Michael A

2016-01-15

Human pluripotent stem cells (PSCs) represent an attractive source of cardiomyocytes with potential applications including disease modeling, drug discovery and safety screening, and novel cell-based cardiac therapies. Insights from embryology have contributed to the development of efficient, reliable methods capable of generating large quantities of human PSC-cardiomyocytes with cardiac purities ranging up to 90%. However, for human PSCs to meet their full potential, the field must identify methods to generate cardiomyocyte populations that are uniform in subtype (e.g. homogeneous ventricular cardiomyocytes) and have more mature structural and functional properties. For in vivo applications, cardiomyocyte production must be highly scalable and clinical grade, and we will need to overcome challenges including graft cell death, immune rejection, arrhythmogenesis, and tumorigenic potential. Here we discuss the types of human PSCs, commonly used methods to guide their differentiation into cardiomyocytes, the phenotype of the resultant cardiomyocytes, and the remaining obstacles to their successful translation. Copyright © 2015 Elsevier B.V. All rights reserved.

NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

PubMed Central

Asano, Masanao; Li, Yue-Sheng; Núñez, Gabriel

2017-01-01

Although B cell development requires expression of the B cell antigen receptor (BCR), it remains unclear whether engagement of self-antigen provides a positive impact for most B cells. Here, we show that BCR engagement by self-ligand during development in vivo results in up-regulation of the Nod-like receptor member Nod1, which recognizes the products of intestinal commensal bacteria. In anti-thymocyte/Thy-1 autoreactive BCR knock-in mice lacking self–Thy-1 ligand, immunoglobulin light chain editing occurred, generating B cells with up-regulated Nod1, including follicular and marginal zone B cells with natural autoreactivity. This BCR editing with increased Nod1 resulted in preferential survival. In normal adult mice, most mature B cells are enriched for Nod1 up-regulated cells, and signaling through Nod1 promotes competitive survival of mature B cells. These findings demonstrate a role for microbial products in promoting survival of mature B cells through up-regulated Nod1, providing a positive effect of BCR engagement on development of most B cells. PMID:28878001

The role of proteinase 3 (PR3) and the protease-activated receptor-2 (PAR-2) pathway in dendritic cell (DC) maturation of human-DC-like monocytes and murine DC.

PubMed

Jiang, Bo; Grage-Griebenow, Evelin; Csernok, Elena; Butherus, Kristine; Ehlers, Stefan; Gross, Wolfgang L; Holle, Julia U

2010-01-01

The aim of the study was to assess PAR-2 expression on dendritic cell (DC) subsets and other immune cells of Wegener's granulomatosis (WG) patients and healthy controls (HC) and to investigate whether Proteinase 3 (PR3, a serine protease which can activate PAR2) induces maturation of human DC-like monocytes and murine Flt-3 ligand- and GM-CSF-generated DC. Human peripheral blood cells including DC subsets and Flt-3l- and GM-CSF-generated mouse DC were analysed for expression of PAR-2 and DC maturation markers by flow cytometry before and after stimulation with PR3, trypsin, PAR-2 agonist or LPS for 24 h. There was no difference of PAR-2 expression on PMNs, monocytes, lymphocytes and DC between all WG samples and HC. However, in inactive WG, expression of PAR-2 was downregulated on the cell surface of PMNs, monocytes, lymphocytes, and CD11c+DC compared to active WG and HC. PR3 and PAR2-agonists did not induce upregulation of PAR-2 or maturation markers of human DC-like monocytes in WG and HC. Likewise, murine PR3 did not induce upregulation of PAR-2 or maturation markers in murine DC. PAR-2 expression is downregulated on human peripheral blood cells including CD11c+ DC in inactive WG compared to active WG and HC, possibly reflecting a non-activated status of these cells in inactive disease. PR3 and PAR-2- agonists did not induce maturation of human ex vivo DC-like monocytes in WG and HC and of murine DC, suggesting this pathway is not singularly involved in the maturation of these cell subsets.

Flavivirus structural heterogeneity: implications for cell entry.

PubMed

Rey, Félix A; Stiasny, Karin; Heinz, Franz X

2017-06-01

The explosive spread of Zika virus is the most recent example of the threat imposed to human health by flaviviruses. High-resolution structures are available for several of these arthropod-borne viruses, revealing alternative icosahedral organizations of immature and mature virions. Incomplete proteolytic maturation, however, results in a cloud of highly heterogeneous mosaic particles. This heterogeneity is further expanded by a dynamic behavior of the viral envelope glycoproteins. The ensemble of heterogeneous and dynamic infectious particles circulating in infected hosts offers a range of alternative possible receptor interaction sites at their surfaces, potentially contributing to the broad flavivirus host-range and variation in tissue tropism. The potential synergy between heterogeneous particles in the circulating cloud thus provides an additional dimension to understand the unanticipated properties of Zika virus in its recent outbreaks. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells.

PubMed

Jung, YunJae

2015-12-01

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 µM dbcAMP or 0.5 µM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 µM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 µM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 µM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

Mature brain-derived neurotrophic factor and its receptor TrkB are upregulated in human glioma tissues.

PubMed

Xiong, Jing; Zhou, L I; Lim, Yoon; Yang, Miao; Zhu, Yu-Hong; Li, Zhi-Wei; Fu, Deng-Li; Zhou, Xin-Fu

2015-07-01

There are two forms of brain-derived neurotrophic factor (BDNF), precursor of BDNF (proBDNF) and mature BDNF, which each exert opposing effects through two different transmembrane receptor signaling systems, consisting of p75 neurotrophin receptor (p75NTR) and tyrosine receptor kinase B (TrkB). Previous studies have demonstrated that proBDNF promotes cell death and inhibits the growth and migration of C6 glioma cells through p75NTR in vitro , while mature BDNF has opposite effects on C6 glioma cells. It is hypothesized that mature BDNF is essential in the development of malignancy in gliomas. However, histological data obtained in previous studies were unable distinguish mature BDNF from proBDNF due to the lack of specific antibodies. The present study investigated the expression of mature BDNF using a specific sheep monoclonal anti-mature BDNF antibody in 42 human glioma tissues of different grades and 10 control tissues. The correlation between mature BDNF and TrkB was analyzed. Mature BDNF expression was significantly increased in high-grade gliomas, and was positively correlated with the malignancy of the tumor and TrkB receptor expression. The present data have demonstrated that increased levels of mature BDNF contribute markedly to the development of malignancy of human gliomas through the primary BDNF receptor TrkB.

Langerhans cell sarcoma following marginal zone lymphoma: expanding the knowledge on mature B cell plasticity.

PubMed

Ambrosio, Maria Raffaella; De Falco, Giulia; Rocca, Bruno Jim; Barone, Aurora; Amato, Teresa; Bellan, Cristiana; Lazzi, Stefano; Leoncini, Lorenzo

2015-10-01

The concept of unidirectional differentiation of the haematopoietic stem cell has been challenged after recent findings that human B cell progenitors and even mature B cells can be reprogrammed into histiocytic/dendritic cells by altering expression of lineage-associated transcription factors. The conversion of mature B cell lymphomas to Langerhans cell neoplasms is not well documented. Three previous reports have described clonally related follicular lymphoma and Langerhans cell tumours, whereas no case has been published of clonally related marginal zone lymphoma and Langerhans cell sarcoma. We describe the case of a 77-year-old patient who developed a Langerhans cell sarcoma and 6 years later a nodal marginal zone lymphoma. Mutation status examination showed 100 % gene identity to the germline sequence, suggesting direct trans-differentiation or dedifferentiation of the nodal marginal zone lymphoma to the Langerhans cell sarcoma rather than a common progenitor. We found inactivation of paired box 5 (PAX-5) in the lymphoma cells by methylation, along with duplication of part of the long arm of chromosomes 16 and 17 in the sarcoma cells. The absence of PAX-5 could have triggered B cells to differentiate into macrophages and dendritic cells. On the other hand, chromosomal imbalances might have activated genes involved in myeloid lineage maturation, transcription activation and oncogenesis. We hypothesize that this occurred because of previous therapies for nodal marginal zone lymphoma. Better understanding of this phenomenon may help in unravelling the molecular interplay between transcription factors during haematopoietic lineage commitment and may expand the spectrum of clonally related mature B cell neoplasms and Langerhans cell tumours.

Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1

PubMed Central

Wu, Gregory F.; Corbo, Evann; Schmidt, Michelle; Smith-Garvin, Jennifer E.; Riese, Matthew J.; Jordan, Martha S.; Laufer, Terri M.; Brown, Eric J.; Maltzman, Jonathan S.

2011-01-01

SUMMARY The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of wild-type SLP-76 protein in thymocytes. Following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T cell mediated autoimmune disease experimental autoimmune encephalomyelitis. Our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects. PMID:21469089

Determination of the Role of Estrogen Receptors and Estrogen Regulated Genes in B cell Autoreactivity. Addendum

DTIC Science & Technology

2012-07-01

Betty Diamond – DOD FINAL REPORT 9 Figure 3: (A) expression of estrogen receptors ERalpha( Esr1 ) and ERbeta (Esr2) in splenic B cells and (B)Urinary...16 OH-Estradiol metabolite in BALB/c and C57BL6 mice. Esr1 0 0.05 0.1 0.15 0.2 Transit. Mature Transit. Mature Transit. Mature Transit. mature P E2 P

Rab10 Disruption Results in Delayed OPC Maturation.

PubMed

Zhang, Zhao-Huan; Zhao, Wei-Qian; Ma, Fan-Fei; Zhang, Hui; Xu, Xiao-Hui

2017-10-01

Oligodendrocyte precursor cell (OPC) maturation requires membrane addition for myelin sheath formation. Since the Rab system has been shown to contribute to membrane addition in other cell types, in this study, we explored the role of Rab in OPC maturation. SiRNA and shRNA techniques and conditional knockout mice provided in vitro and in vivo evidence that Rab10 is involved in OPC maturation and may affect myelination during OPC development.

Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor

PubMed Central

Fujimoto, Michiko; Hayashi, Teruo; Urfer, Roman; Mita, Shiro; Su, Tsung-Ping

2013-01-01

The sigma-1 receptor (Sig-1R) is a novel endoplasmic reticulum (ER) molecular chaperone that regulates protein folding and degradation. The Sig-1R activation by agonists is known to improve memory, promote cell survival, and exert an antidepressant-like action in animals. Cutamesine (SA4503), a selective Sig-1R ligand, was shown to increase BDNF in the hippocampus of rats. How exactly the intracellular chaperone Sig-1R or associated ligand causes the increase of BDNF or any other neurotrophins is unknown. We examined here whether the action of Sig-1Rs may relate to the post-translational processing and release of BDNF in neuroblastoma cell lines. We used in vitro assays and confirmed that cutamesine possesses the bona fide Sig-1R agonist property by causing the dissociation of BiP from Sig-1Rs. The C-terminus of Sig-1Rs exerted robust chaperone activity by completely blocking the aggregation of BDNF and GDNF in vitro. Chronic treatment with cutamesine in rat B104 neuroblastoma caused a time- and dose-dependent potentiation of the secretion of BDNF without affecting the mRNA level of BDNF. Cutamesine decreased the intracellular level of pro-BDNF and mature BDNF whereas increased the extracellular level of mature BDNF. The pulse-chase experiment indicated that the knockdown of Sig-1Rs decreased the secreted mature BDNF in B104 cells without affecting the synthesis of BDNF. Our findings indicate that, in contrast to clinically used antidepressants that promote the transcriptional upregulation of BDNF, the Sig-1R agonist cutamesine potentiates the post-translational processing of neurotrophins. This unique pharmacological profile may provide a novel therapeutic opportunity for the treatment of neuropsychiatric disorders. PMID:22337473

Generation of functional hepatocytes from human spermatogonial stem cells.

PubMed

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-02-23

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration.

Generation of functional hepatocytes from human spermatogonial stem cells

PubMed Central

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-01-01

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

Stem Cells Derived from Tooth Periodontal Ligament Enhance Functional Angiogenesis by Endothelial Cells

PubMed Central

Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J.; Tarle, Susan A.

2014-01-01

In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential, which could make them a very attractive cell population for utilization in regenerative cell therapies. PMID:24147894

B-Cell Maturation Antigen, A Proliferation-Inducing Ligand, and B-Cell Activating Factor Are Candidate Mediators of Spinal Cord Injury-Induced Autoimmunity

PubMed Central

Saltzman, Jonah W.; Battaglino, Ricardo A.; Salles, Loise; Jha, Prateek; Sudhakar, Supreetha; Garshick, Eric; Stott, Helen L.; Zafonte, Ross

2013-01-01

Abstract Autoimmunity is thought to contribute to poor neurological outcomes after spinal cord injury (SCI). There are few mechanism-based therapies, however, designed to reduce tissue damage and neurotoxicity after SCI because the molecular and cellular bases for SCI-induced autoimmunity are not completely understood. Recent groundbreaking studies in rodents indicate that B cells are responsible for SCI-induced autoimmunity. This novel paradigm, if confirmed in humans, could aid in the design of neuroprotective immunotherapies. The aim of this study was to investigate the molecular signaling pathways and mechanisms by which autoimmunity is induced after SCI, with the goal of identifying potential targets in therapies designed to reduce tissue damage and inflammation in the chronic phase of SCI. To that end, we performed an exploratory microarray analysis of peripheral blood mononuclear cells to identify differentially expressed genes in chronic SCI. We identified a gene network associated with lymphoid tissue structure and development that was composed of 29 distinct molecules and five protein complexes, including two cytokines, a proliferation-inducing ligand (APRIL) and B-cell–activating factor (BAFF), and one receptor, B-cell maturation antigen (BMCA) involved in B cell development, proliferation, activation, and survival. Real-time polymerase chain reaction analysis from ribonucleic acid samples confirmed upregulation of these three genes in SCI. To our knowledge, this is the first report that peripheral blood mononuclear cells produce increased levels of BAFF and APRIL in chronic SCI. This finding provides evidence of systemic regulation of SCI-autoimmunity via APRIL and BAFF mediated activation of B cells through BMCA and points toward these molecules as potential targets of therapies designed to reduce neuroinflammation after SCI. PMID:23088438

BCR mediated signal transduction in immature and mature B cells.

PubMed

Koncz, Gábor; Bodor, Csaba; Kövesdi, Dorottya; Gáti, Róbert; Sármay, Gabriella

2002-06-03

Ligation of B cell receptors (BCR) on immature B cells may induce apoptosis, while in mature B cells it stimulates cell activation and growth. The signaling pathway regulating the differential functional response, death or survival of the B cell is not fully characterized. We have tested the intracellular signaling requirement of these processes using B cells isolated from the spleen of irradiated auto-reconstituted (transitional immature B cells) and untreated mice (mature B cells), respectively. We compared the BCR induced intracellular [Ca2+] transient, protein tyrosine phosphorylation and ERK phosphorylation, furthermore, the activation of Elk-1 and CREB transcription factors. The BCR induced rise of intracellular [Ca2+] did not significantly differ in the two populations, only a slight difference in the late phase of the response was observed. Immature B cells responded with a maximum tyrosine phosphorylation to a five times lower dose of anti-IgM compared to the mature population. Most importantly, we have found a significant difference in the tyrosine phosphorylation of the Gab family adaptor proteins, Gab1/2. In contrast to mature B cells, crosslinking of BCR on immature B cells did not induce tyrosine phosphorylation of Gab2, thus the Gab2-organized signal amplification complex could not be produced. Furthermore, we detected a significant difference in the kinetics of BCR induced ERK, Elk-1 and CREB phosphorylation. In immature B cells, ERK was transiently phosphorylated, ceasing after 120 min, while in mature cells, ERK phosphorylation was sustained. Elk-1 and CREB activation was also transient in immature B cells, followed the kinetics of ERK phosphorylation. The lack of sustained Erk1/2 activation suppresses the transcription factors necessary for the proliferation signal. Since ERK is regulated by the phosphorylated Gab1/2, these data demonstrate that BCR triggered phosphorylation and signal amplification of Gab1/2 is a critical step in a life or death decision of B cells.

Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

PubMed

Polański, Zbigniew; Homer, Hayden; Kubiak, Jacek Z

2012-01-01

Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

A Novel Source of Cultured Podocytes

PubMed Central

Da Sacco, Stefano; Lemley, Kevin V.; Sedrakyan, Sargis; Zanusso, Ilenia; Petrosyan, Astgik; Peti-Peterdi, Janos; Burford, James; De Filippo, Roger E.; Perin, Laura

2013-01-01

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies. PMID:24349133

Tumor necrosis factor-{alpha} enhances IL-15-induced natural killer cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jiwon; Lee, Suk Hyung; Korea University of Science and Technology, Yusong, Daejeon 305-333

2009-09-04

The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-{alpha} (TNF-{alpha}) is a positive regulator of NK cell differentiation. TNF-{alpha} augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-{alpha} alone also induced NK cell maturation as well as IL-15. TNF-{alpha} also increased IFN-{gamma} production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-{alpha} and IL-15. In addition, TNF-{alpha} increased nuclear factor-kappamore » B (NF-{kappa}B) activity in NK cells and inhibition of NF-{kappa}B impeded TNF-{alpha}-enhanced NK cell maturation. Overall, these data suggest that TNF-{alpha} significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-{kappa}B activity.« less

Progranulin, a Major Secreted Protein of Mouse Adipose-Derived Stem Cells, Inhibits Light-Induced Retinal Degeneration

PubMed Central

Tsuruma, Kazuhiro; Yamauchi, Mika; Sugitani, Sou; Otsuka, Tomohiro; Ohno, Yuta; Nagahara, Yuki; Ikegame, Yuka; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru

2014-01-01

Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose-derived stem cells (ASCs) and ASC-conditioned medium (ASC-CM) against retinal damage and identified the neuroprotective factors in ASC-CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light-induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC-CM and mature adipocyte-conditioned medium were collected after 72 hours of culture. In vitro, H2O2- and light-induced cell death was reduced in a photoreceptor cell line with ASC-CM but not with mature adipocyte-conditioned medium. In vivo, light-induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC-CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal-regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC-CM and progranulin have neuroprotective effects in the light-induced retinal-damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina. PMID:24233842

Progranulin, a major secreted protein of mouse adipose-derived stem cells, inhibits light-induced retinal degeneration.

PubMed

Tsuruma, Kazuhiro; Yamauchi, Mika; Sugitani, Sou; Otsuka, Tomohiro; Ohno, Yuta; Nagahara, Yuki; Ikegame, Yuka; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru; Hara, Hideaki

2014-01-01

Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose-derived stem cells (ASCs) and ASC-conditioned medium (ASC-CM) against retinal damage and identified the neuroprotective factors in ASC-CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light-induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC-CM and mature adipocyte-conditioned medium were collected after 72 hours of culture. In vitro, H2O2- and light-induced cell death was reduced in a photoreceptor cell line with ASC-CM but not with mature adipocyte-conditioned medium. In vivo, light-induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC-CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal-regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC-CM and progranulin have neuroprotective effects in the light-induced retinal-damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.

Rescue of the mature B cell compartment in BAFF-deficient mice by treatment with recombinant Fc-BAFF.

PubMed

Swee, Lee Kim; Tardivel, Aubry; Schneider, Pascal; Rolink, Antonius

2010-06-15

BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Alpha-fetoprotein, stem cells and cancer: how study of the production of alpha-fetoprotein during chemical hepatocarcinogenesis led to reaffirmation of the stem cell theory of cancer.

PubMed

Sell, Stewart

2008-01-01

Identification of the cells in the liver that produce alpha-fetoprotein during development, in response to liver injury and during the early stages of chemical hepatocarcinogenesis led to the conclusion that maturation arrest of liver-determined tissue stem cells was the cellular process that gives rise to hepatocellular carcinomas. When the cellular changes in these processes were compared to that of the formation of teratocarcinomas, the hypothesis arose that all cancers arise from maturation arrest of tissue-determined stem cells. This was essentially a reinterpretation of the embryonal rest theory of cancer whereby tissue stem cells take the role of embryonal rests. A corollary of the stem cell theory of the origin of cancer is that cancers contain the same functional cell populations as normal tissues: stem cells, transit-amplifying cells and mature cells. Cancer stem cells retain the essential feature of normal stem cells: the ability to self-renew. Growth of cancers is due to continued proliferation of cancer transit-amplifying cells that do not differentiate to mature cells (maturation arrest). On the other hand, cancer stem cells generally divide very rarely and contribute little to tumor growth. However, the presence of cancer stem cells in tumors is believed to be responsible for the properties of immortalization, transplantability and resistance to therapy characteristic of cancers. Current therapies for cancer (chemotherapy, radiotherapy, antiangiogenesis and differentiation therapy) are directed against the cancer transit-amplifying cells. When these therapies are discontinued, the cancer reforms from the cancer stem cells. Therapy directed toward interruption of the cell signaling pathways that maintain cancer stem cells could lead to new modalities to the prevention of regrowth of the cancer. Copyright 2008 S. Karger AG, Basel.

ALPHA-FETOPROTEIN (AFP), STEM CELLS, AND CANCER: HOW STUDY OF THE PRODUCTION OF AFP DURING CHEMICAL HEPATOCARCINOGENESIS LED TO REAFFIRMATION OF THE STEM CELL THEORY OF CANCER

PubMed Central

Sell, Stewart

2008-01-01

Identification of the cells in the liver that produce alpha-fetoprotein (AFP) during development, in response to liver injury, and during the early stages of chemical hepatocarcinogenesis led to the conclusion that maturation arrest of liver-determined tissue stem cells was the cellular process that gives rise to hepatocellular carcinomas (HCC). When the cellular changes in these processes were compared that of the formation of teratocarcinomas, the hypothesis arose that all cancers arise from maturation arrest of tissue determined stem cells. This was essentially a reinterpretation of the embryonal rest theory of cancer whereby tissue stem cells take the role of embryonal rests. A corollary of the stem cell theory of the origin of cancer is that cancers contain the same functional cell populations as do normal tissues: stem cells, transit-amplifying cells, and mature cells. Cancer stem cells retain the essential feature of normal stem cells: the ability to self-renew. Growth of cancers is due to continued proliferation of cancer transit-amplifying cells that do not differentiate to mature cells (maturation arrest). On the other hand, cancer stem cells generally divide very rarely and contribute little to tumor growth. However, the presence of cancer stem cells in tumors is believed to be responsible for the properties of immortalization, transplantability and resistance to therapy characteristic of cancers. Current therapies for cancer (chemotherapy, radiotherapy, anti-angiogenesis and differentiation therapy) are directed against the cancer transit amplifying cells. When these therapies are discontinued, the cancer re-forms from the cancer stem cells. Therapy directed toward interruption of the cell-signaling pathways that maintain cancer stem cells could lead to new modalities to the prevention of re-growth of the cancer. PMID:18612221

Comparative morphology and ultrastructure of the prosomal salivary glands in the unfed larvae Leptotrombidium orientale (Acariformes, Trombiculidae), a possible vector of tsutsugamushi disease agent.

PubMed

Shatrov, Andrew B

2015-07-01

The prosomal salivary glands of the unfed larvae Leptotrombidium orientale (Schluger) were investigated using transmission electron microscopy. In total, four pairs of the prosomal glands were identified--three pairs, the lateral, the medial and the anterior, belong to the podocephalic system, and one pair, the posterior, is separate having an own excretory duct. All glands are simple alveolar/acinous with prismatic cells arranged around a relatively small intra-alveolar lumen with the duct base. The cells of all glands besides the lateral ones contain practically mature electron-dense secretory granules ready to be discharged from the cells. The secretory granules in the lateral glands undergo formation and maturation due to the Golgi body activity. The cells of all gland types contain a large basally located nucleus and variously expressed rough endoplasmic reticulum. Specialized duct-forming cells filled with numerous freely scattered microtubules are situated in the middle zone of each gland's acinus and form the intra-alveolar lumen and the duct base. Both the acinar (secretory) and the duct-forming cells contact each other via gap junctions and septate desmosomes. Axons of nerve cells come close to the basal extensions of the duct-forming cells where they form the bulb-shaped synaptic terminations. The process of secretion is under the control of the nerve system that provides contraction of the duct-forming cells and discharge of secretion from the secretory cells into the intra-alveolar lumen and further to the exterior. Unfed larvae of L. orientale, the potential vector of tsutsugamushi disease agents, contain the most simply organized salivary secretory granules among known trombiculid larvae, and this secretion, besides the lateral glands, does not undergo significant additional maturation. Thus, the larvae are apparently ready to feed on the appropriate host just nearly after hatching.

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of Carica papaya.

PubMed

Vale, Ellen Moura; Reis, Ricardo Souza; Passamani, Lucas Zanchetta; Santa-Catarina, Claudete; Silveira, Vanildo

2018-03-01

Efficient protocols for somatic embryogenesis of papaya ( Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C . papaya . To study the effects of PEG on somatic embryogenesis of C . papaya , we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG (control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C . papaya somatic embryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.

Rift Valley fever virus incorporates the 78kDa glycoprotein into virions matured in C6/36 2 mosquito cells

USDA-ARS?s Scientific Manuscript database

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment codi...

Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.

PubMed

Johnston, Simon A; May, Robin C

2013-03-01

Cryptococcus is a potentially fatal fungal pathogen and a leading cause of death in immunocompromised patients. As an opportunistic and facultative intracellular pathogen of humans, Cryptococcus exhibits a complex set of interactions with the host immune system in general, and macrophages in particular. Cryptococcus is resistant to phagocytosis but is also able to survive and proliferate within the mature phagolysosome. It can cause the lysis of host cells, can be transferred between macrophages or exit non-lytically via vomocytosis. Efficient phagocytosis is reliant on opsonization and Cryptococcus has a number of anti-phagocytic strategies including formation of titan cells and a thick polysaccharide capsule. Following uptake, phagosome maturation appears to occur normally, but the internalized pathogen is able to survive and replicate. Here we review the interactions and host manipulation processes that occur within cryptococcal-infected macrophages and highlight areas for future research. © 2012 Blackwell Publishing Ltd.

Solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation: piece by piece.

PubMed

Lundy, David J; Lee, Desy S; Hsieh, Patrick C H

2017-03-01

There is a growing need for in vitro models which can serve as platforms for drug screening and basic research. Human adult cardiomyocytes cannot be readily obtained or cultured, and so pluripotent stem cell-derived cardiomyocytes appear to be an attractive option. Unfortunately, these cells are structurally and functionally immature-more comparable to foetal cardiomyocytes than adult. A recent study by Ruan et al ., provides new insights into accelerating the maturation process and takes us a step closer to solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation.

New peptide MY1340 revert the inhibition effect of VEGF on dendritic cells differentiation and maturation via blocking VEGF-NRP-1 axis and inhibit tumor growth in vivo.

PubMed

Mo, Zheng; Yu, Fei; Han, Su; Yang, Songhua; Wu, Liangliang; Li, Peng; Jiao, Shunchang

2018-05-03

The development and clinical application of immunostimulatory therapy provides us a new and exciting strategy in cancer treatment of which the agents act on crucial receptors. Given the fact that Neuropilin-1(NRP-1) is essential for vascular endothelial growth factor (VEGF) to inhibit LPS-dependent maturation of dendritic cells (DCs), it may present a potentially meaningful target in cancer immunotherapy. To explore this hypothesis, we synthesized a novel polypeptide called MY1340 consist of 32 amino acids with the aim of targeting VEGF-NRP-1 axis. Pull-down assay coupled with liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) was firstly conducted to identify NRP-1 as a potential MY1340 interacting protein, and the interaction between them was further confirmed by western blot. The competitive enzyme-linked immunosorbent assay (ELISA) results revealed that MY1340 was able to inhibit the binding between NRP-1 and VEGF with IC 50 7.42 ng/ml, better than that of Tuftsin, although a natural ligand reportedly specific for the NRP-1 receptor. The presence of VEGF significantly reduced the expression of human leukocyte antigen-DR (HLA-DR), CD86 and CD11C on DCs, and this effect was reverted by MY1340-augment p65 NF-κB and ERK1/2 phosphorylation. We also present evidence that MY1340 is remarkably efficacious in the treatment of mice bearing subcutaneous liver cancer and induced DC maturation in the tumor environment in vivo. Taken together, these results indicate that MY1340 may represent a potential efficient immune therapeutic compound within disease that are rich in VEGF. Copyright © 2018 Elsevier B.V. All rights reserved.

Differentiation and Characterization of Myeloid Cells

PubMed Central

Gupta, Dipti; Shah, Hetavi Parag; Malu, Krishnakumar; Berliner, Nancy; Gaines, Peter

2015-01-01

Recent molecular studies of myeloid differentiation have utilized several in vitro models of myelopoiesis, generated from either ex vivo differentiated bone marrow progenitors or induced immortalized myeloid cell lines. Ex vivo differentiation begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34+ antigen (human) or Sca-1+ (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid–induced myeloid development, however, both cell lines exhibit defects in the upregulation of late-expressed neutrophil-specific genes. Multiple murine factor–dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G-CSF-induced myeloid maturation, EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid, and ER-Hoxb8 cells, which undergo myeloid maturation upon removal of estradial in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT-PCR. Together, these assays provide a solid foundation for in vitro investigations of myeloid development with either human or mouse models. PMID:24510620

Erythrocyte Enrichment in Hematopoietic Progenitor Cell Cultures Based on Magnetic Susceptibility of the Hemoglobin

PubMed Central

Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V.; Moore, Lee R.; Chalmers, Jeffrey J.; Zborowski, Maciej

2012-01-01

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. PMID:22952572

Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.

PubMed

Young, Kira; Borikar, Sneha; Bell, Rebecca; Kuffler, Lauren; Philip, Vivek; Trowbridge, Jennifer J

2016-10-17

Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging. © 2016 Young et al.

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung.

PubMed

Lange, Alexander W; Sridharan, Anusha; Xu, Yan; Stripp, Barry R; Perl, Anne-Karina; Whitsett, Jeffrey A

2015-02-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

PubMed

Leon, P M M; Campos, V F; Kaefer, C; Begnini, K R; McBride, A J A; Dellagostin, O A; Seixas, F K; Deschamps, J C; Collares, T

2013-08-01

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

The active translation of MHCII mRNA during dendritic cells maturation supplies new molecules to the cell surface pool.

PubMed

Malanga, Donatella; Barba, Pasquale; Harris, Paul E; Maffei, Antonella; Del Pozzo, Giovanna

2007-04-01

The transition of human dendritic cells (DCs) from the immature to the mature phenotype is characterized by an increased density of MHC class II (MHCII) molecules on the plasma membrane, a key requirement of their competence as professional antigen presenting cells (APCs). MHCII molecules on the cell surface derive from newly synthesized as well as from preexisting proteins. So far, all the studies done on DCs during maturation, to establish the relative contribution of newly synthesized MHCII molecules to the cell surface pool did not produced a clear, unified scenario. We report that, in human DCs stimulated ex vivo with LPS, the changes in the RNA accumulation specific for at least two MHCII genes (HLA-DRA and HLA-DQA1) due to transcriptional upregulation, is associated with the active translation at high rate of these transcripts. Our finding reveals that, across the 24h of the maturation process in human DCs, newly synthesized MHCII proteins are supplied to the APCs cell surface pool.

Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application.

PubMed

Saito, Shigeo; Lin, Ying-Chu; Murayama, Yoshinobu; Nakamura, Yukio; Eckner, Richard; Niemann, Heiner; Yokoyama, Kazunari K

2015-12-01

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment.

Efficient generation of hPSC-derived midbrain dopaminergic neurons in a fully defined, scalable, 3D biomaterial platform

PubMed Central

Adil, Maroof M.; Rodrigues, Gonçalo M. C.; Kulkarni, Rishikesh U.; Rao, Antara T.; Chernavsky, Nicole E.; Miller, Evan W.; Schaffer, David V.

2017-01-01

Pluripotent stem cells (PSCs) have major potential as an unlimited source of functional cells for many biomedical applications; however, the development of cell manufacturing systems to enable this promise faces many challenges. For example, there have been major recent advances in the generation of midbrain dopaminergic (mDA) neurons from stem cells for Parkinson’s Disease (PD) therapy; however, production of these cells typically involves undefined components and difficult to scale 2D culture formats. Here, we used a fully defined, 3D, thermoresponsive biomaterial platform to rapidly generate large numbers of action-potential firing mDA neurons after 25 days of differentiation (~40% tyrosine hydroxylase (TH) positive, maturing into 25% cells exhibiting mDA neuron-like spiking behavior). Importantly, mDA neurons generated in 3D exhibited a 30-fold increase in viability upon implantation into rat striatum compared to neurons generated on 2D, consistent with the elevated expression of survival markers FOXA2 and EN1 in 3D. A defined, scalable, and resource-efficient cell culture platform can thus rapidly generate high quality differentiated cells, both neurons and potentially other cell types, with strong potential to accelerate both basic and translational research. PMID:28091566

Probiotic metabolites from Bacillus coagulans GanedenBC30TM support maturation of antigen-presenting cells in vitro

PubMed Central

Benson, Kathleen F; Redman, Kimberlee A; Carter, Steve G; Keller, David; Farmer, Sean; Endres, John R; Jensen, Gitte S

2012-01-01

AIM: To study the effects of probiotic metabolites on maturation stage of antigen-presenting immune cells. METHODS: Ganeden Bacillus coagulans 30 (GBC30) bacterial cultures in log phase were used to isolate the secreted metabolite (MET) fraction. A second fraction was made to generate a crude cell-wall-enriched fraction, by centrifugation and lysis, followed by washing. A preparation of MET was subjected to size exclusion centrifugation, generating three fractions: < 3 kDa, 3-30 kDa, and 30-200 kDa and activities were tested in comparison to crude MET and cell wall in primary cultures of human peripheral blood mononuclear cell (PBMC) as a source of antigen-presenting mononuclear phagocytes. The maturation status of mononuclear phagocytes was evaluated by staining with monoclonal antibodies towards CD14, CD16, CD80 and CD86 and analyzed by flow cytometry. RESULTS: Treatment of PBMC with MET supported maturation of mononuclear phagocytes toward both macrophage and dendritic cell phenotypes. The biological activity unique to the metabolites included a reduction of CD14+ CD16+ pro-inflammatory cells, and this property was associated with the high molecular weight metabolite fraction. Changes were also seen for the dendritic cell maturation markers CD80 and CD86. On CD14dim cells, an increase in both CD80 and CD86 expression was seen, in contrast to a selective increase in CD86 expression on CD14bright cells. The co-expression of CD80 and CD86 indicates effective antigen presentation to T cells and support of T helper cell differentiation. The selective expression of CD86 in the absence of CD80 points to a role in generating T regulatory cells. CONCLUSION: The data show that a primary mechanism of action of GBC30 metabolites involves support of more mature phenotypes of antigen-presenting cells, important for immunological decision-making. PMID:22563167

Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization

PubMed Central

Nakshatri, Harikrishna; Anjanappa, Manjushree; Bhat-Nakshatri, Poornima

2015-01-01

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations. PMID:26311223

Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization.

PubMed

Nakshatri, Harikrishna; Anjanappa, Manjushree; Bhat-Nakshatri, Poornima

2015-08-27

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations.

The impact of fruit maturation on bioactive microconstituents, inhibition of serum oxidation and inflammatory markers in stimulated PBMCs and sensory characteristics of Koroneiki virgin olive oils from Messenia, Greece.

PubMed

Kaliora, Andriana C; Artemiou, Anna; Giogios, Ioannis; Kalogeropoulos, Nick

2013-08-01

Olive fruits from the Koroneiki cultivar (Olea europaea L.) grown in Messenia, Greece, were hand-picked from the same trees in progressive maturity stages, covering three months, and processed identically with a commercial olive mill and a three-phase decanter. Data on quality parameters, and antioxidant activity of the obtained oils were collected by employing the conventional analytical methods set by European Union Commission Regulation no. 61/2011. Additionally, the potential of oils' polar extract to inhibit total serum lipid oxidation and inflammatory markers in stimulated human mononuclear cells was assayed. The results showed that ripening caused an increase in monounsaturated and decrease in polyunsaturated fatty acids, as well as an increase in phenolic compounds - mainly hydroxytyrosol - and in squalene. The extracts' ferric reducing power was in line with the increase of phenolic compounds. In later stages of maturation, lipoprotein oxidation was less potent and the decrease of inflammatory markers in stimulated human mononuclear cells was more powerful. Sensory evaluation detected differences in oils' "bitter" attributes, while the analysis of oils' volatiles revealed quantitative differences.

Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.

PubMed

Rezania, Alireza; Bruin, Jennifer E; Xu, Jean; Narayan, Kavitha; Fox, Jessica K; O'Neil, John J; Kieffer, Timothy J

2013-11-01

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs, composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive), endocrine precursors (NKX2.2/synaptophysin-positive, hormone/NKX6.1-negative), and polyhormonal cells (insulin/glucagon-positive, NKX6.1-negative). However, the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question, we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant, both populations contained a high proportion of PDX1-expressing cells (~85%-90%) but were distinguished by their relatively high (~80%) or low (~25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study, but only NKX6.1-high grafts displayed robust meal-, glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue, but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells, whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high, but not NKX6.1-low grafts expressed nuclear MAFA. Collectively, this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional β-cells with only a minor contribution from the endocrine subpopulation. © AlphaMed Press.

Rax Homeoprotein Regulates Photoreceptor Cell Maturation and Survival in Association with Crx in the Postnatal Mouse Retina.

PubMed

Irie, Shoichi; Sanuki, Rikako; Muranishi, Yuki; Kato, Kimiko; Chaya, Taro; Furukawa, Takahisa

2015-08-01

The Rax homeobox gene plays essential roles in multiple processes of vertebrate retina development. Many vertebrate species possess Rax and Rax2 genes, and different functions have been suggested. In contrast, mice contain a single Rax gene, and its functional roles in late retinal development are still unclear. To clarify mouse Rax function in postnatal photoreceptor development and maintenance, we generated conditional knockout mice in which Rax in maturing or mature photoreceptor cells was inactivated by tamoxifen treatment (Rax iCKO mice). When Rax was inactivated in postnatal Rax iCKO mice, developing photoreceptor cells showed a significant decrease in the level of the expression of rod and cone photoreceptor genes and mature adult photoreceptors exhibited a specific decrease in cone cell numbers. In luciferase assays, we found that Rax and Crx cooperatively transactivate Rhodopsin and cone opsin promoters and that an optimum Rax expression level to transactivate photoreceptor gene expression exists. Furthermore, Rax and Crx colocalized in maturing photoreceptor cells, and their coimmunoprecipitation was observed in cultured cells. Taken together, these results suggest that Rax plays essential roles in the maturation of both cones and rods and in the survival of cones by regulating photoreceptor gene expression with Crx in the postnatal mouse retina. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

PD-1(HIGH) Follicular CD4 T Helper Cell Subsets Residing in Lymph Node Germinal Centers Correlate with B Cell Maturation and IgG Production in Rhesus Macaques.

PubMed

Xu, Huanbin; Wang, Xiaolei; Lackner, Andrew A; Veazey, Ronald S

2014-01-01

CD4+ T follicular helper (TFH) cells guide development and maturation of B cells and are crucial for effective antibody responses. Here we found rhesus macaque TFH cells, defined as CXCR5+CD4 T cells, contain two major populations: PD-1(INT) and PD-1(HIGH) cells. Of these, PD-1(HIGH)CD4+ T cells highly co-express ICOS but little CCR7, and reside in lymph node germinal centers (GCs), but not in blood. These cells secrete IL-21 and express transcriptional factor Bcl-6 at higher levels than CXCR5+PD-1(INT)CD4+ T cells. In addition, the frequency of PD-1(HIGH)CD4+ T cells is low in lymph nodes of newborns, but increases with age. Levels of PD-1(HIGH)CD4+ T cells correlate with mature B cells in lymph nodes, and PD-1 blockade in PD-1(HIGH)CD4+ T and B cell co-cultures significantly inhibits IgG production. In summary, PD-1(HIGH)CD4+ T cells residing in GC represent a specific TFH subset that contributes to maturation of B cells and IgG production.

Mycobacterium avium subspecies impair dendritic cell maturation.

PubMed

Basler, Tina; Brumshagen, Christina; Beineke, Andreas; Goethe, Ralph; Bäumer, Wolfgang

2013-10-01

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic, granulomatous enteritis of ruminants. Dendritic cells (DC) of the gut are ideally placed to combat invading mycobacteria; however, little is known about their interaction with MAP. Here, we investigated the interaction of MAP and the closely related M. avium ssp. avium (MAA) with murine DC and the effect of infected macrophages on DC maturation. The infection of DC with MAP or MAA induced DC maturation, which differed to that of LPS as maturation was accompanied by higher production of IL-10 and lower production of IL-12. Treatment of maturing DC with supernatants from mycobacteria-infected macrophages resulted in impaired DC maturation, leading to a semi-mature, tolerogenic DC phenotype expressing low levels of MHCII, CD86 and TNF-α after LPS stimulation. Though the cells were not completely differentiated they responded with an increased IL-10 and a decreased IL-12 production. Using recombinant cytokines we provide evidence that the semi-mature DC phenotype results from a combination of secreted cytokines and released antigenic mycobacterial components of the infected macrophage. Our results indicate that MAP and MAA are able to subvert DC function directly by infecting and indirectly via the milieu created by infected macrophages.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

PubMed

Xiong, X R; Lan, D L; Li, J; Zi, X D; Li, M Y

2016-12-01

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division. © 2016 Blackwell Verlag GmbH.

Heterogeneity of Clonal Expansion and Maturation-Linked Mutation Acquisition in Hematopoietic Progenitors in Human Acute Myeloid Leukemia

PubMed Central

Walter, Roland B.; Laszlo, George S.; Lionberger, Jack M.; Pollard, Jessica A.; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Othus, Megan; Rafii, Shahin; Meshinchi, Soheil; Appelbaum, Frederick R.; Bernstein, Irwin D.

2014-01-01

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML) but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition, and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34+/CD33− cells revealed polyclonal growth in highly curable AMLs, suggesting mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34+/CD33− cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications. PMID:24721792

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

PubMed Central

2015-01-01

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 µM dbcAMP or 0.5 µM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 µM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 µM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 µM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity. PMID:26770185

Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle.

PubMed

Wongsrikeao, Pimprapar; Nagai, Takashi; Agung, Budiyanto; Taniguchi, Masayasu; Kunishi, Miho; Suto, Shizuyo; Otoi, Takeshige

2007-06-01

The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos. (c) 2006 Wiley-Liss, Inc.

Frequent homozygosity in both mature and immature ovarian teratomas: a shared genetic basis of tumorigenesis.

PubMed

Snir, Olivia L; DeJoseph, Maura; Wong, Serena; Buza, Natalia; Hui, Pei

2017-10-01

Although homozygosity is well documented in mature teratomas, the genetic zygosity of ovarian immature teratomas and mixed germ cell tumors is less well studied. Ten cases of mature cystic teratomas, eleven cases of grade 2 or 3 immature teratomas, and seven cases of mixed germ cell tumors with an immature teratoma component were investigated by short tandem repeat genotyping to interrogate their genetic zygosity. DNA genotyping was informative in eight mature teratomas, seven immature teratomas and six cases of mixed germ cell tumors. Out of the eight mature teratomas, five cases showed partial or complete homozygosity (63%) with two cases demonstrating complete homozygosity (25%). Of the immature teratomas, six cases showed partial or complete homozygosity (86%) with two cases demonstrating complete homozygosity (29%). For the mixed germ cell tumors, two cases showed partial homozygosity (33%) and none displayed complete homozygosity. Long-term clinical follow-up was available for five immature teratomas (mean follow-up 110 months) and five mixed germ cell tumors (mean follow-up 66 months). None of the five patients with pure immature teratoma had a recurrence; in contrast, four out of five mixed ovarian germ cell tumors recurred between 4 months to 8 years (P=0.048). In conclusion, both immature and mature teratomas harbor frequent genetic homozygosity suggesting a common cellular origin involving germ cells at the same developmental stage. The difference in the rate of homozygosity and tumor recurrence between pure immature teratomas and mixed germ cell tumors suggests that the two entities may involve different pathogenetic pathways and likely pursue different biological behaviors.

Multipotent versus differentiated cell fate selection in the developing Drosophila airways

PubMed Central

Matsuda, Ryo; Hosono, Chie; Samakovlis, Christos; Saigo, Kaoru

2015-01-01

Developmental potentials of cells are tightly controlled at multiple levels. The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells (P-fate) and a distally located population of more differentiated cells (D-fate). We show that the GATA-family transcription factor (TF) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless (Vvl/Drifter/U-turned) stimulates the D-fate. Hedgehog and receptor tyrosine kinase (RTK) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification. Local concentrations of Decapentaplegic/BMP, Wingless/Wnt, and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials, which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway. DOI: http://dx.doi.org/10.7554/eLife.09646.001 PMID:26633813

Properties of a Novel pH-dependent Ca2+ Permeation Pathway Present in Male Germ Cells with Possible Roles in Spermatogenesis and Mature Sperm Function

PubMed Central

Santi, Celia M.; Santos, Teresa; Hernández-Cruz, Arturo; Darszon, Alberto

1998-01-01

Rises of intracellular Ca2+ ([Ca2+]i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca2+]i and intracellular pH (pHi) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca2+ channels modulated by pHi in the origin of these Ca2+ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca2+]i on identified spermatogenic cells from mouse adult testes. In BCECF [(2′,7′)-bis(carboxymethyl)- (5,6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30–60 s) application of 25 mM NH4Cl increased pHi by ∼1.3 U from a resting pHi ∼6.65. A steady pHi plateau was maintained during NH4Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca2+]i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca2+ influx or intracellular Ca2+ release demonstrated that the majority of the Ca2+ rise results from plasma membrane Ca2+ influx, although a small component likely to result from intracellular Ca2+ release was occasionally observed. Ca2+ transients potentiated with repeated NH4Cl applications, gradually obliterating the initial [Ca2+]i drop. The pH-sensitive Ca2+ permeation pathway allows the passage of other divalents (Sr2+, Ba2+, and Mn2+) and is blocked by inorganic Ca2+ channel blockers (Ni2+ and Cd2+), but not by the organic blocker nifedipine. The magnitude of these Ca2+ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca2+ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca2+ channel also present in spermatogenic cells. We postulate that the Ca2+ permeation pathway regulated by pHi, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca2+ influx required for initiating the acrosome reaction and perhaps other important sperm functions. PMID:9649582

BCR-crosslinking induces a transcription of protein phosphatase component G5PR that is required for mature B-cell survival

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huq Ronny, Faisal Mahmudul; Igarashi, Hideya; Core Research for Evolutional Science and Technology

2006-02-03

BCR-crosslinking triggers activation-induced cell death (AICD) selectively in the restricted stage of B-cell differentiation. We examined the transcription of a protein phosphatase subunit G5PR in immature and mature B-cells, because absence of this factor augmented cell sensitivity to AICD, associated with increased activation of JNK and Bim. BCR-crosslinking-induced G5pr transcription in AICD-resistant mature splenic IgM{sup lo}IgD{sup hi} B-cells but not in AICD susceptible immature IgM{sup hi}IgD{sup lo} B-cells. Thus, G5pr induction correlated with the prevention of AICD; High in mature splenic CD23{sup hi} B-cells but low in immature B-cells of neonatal mice, sub-lethally irradiated mice, or xid mice. Lack ofmore » G5pr upregulation was associated with the prolonged activation of JNK. The G5pr cDNA transfection protected an immature B-cell line WEHI-231 from BCR-mediated AICD. The differential expression of G5PR might be responsible for the antigen-dependent selection of B-cells.« less

Influence and timing of arrival of murine neural crest on pancreatic beta cell development and maturation.

PubMed

Plank, Jennifer L; Mundell, Nathan A; Frist, Audrey Y; LeGrone, Alison W; Kim, Thomas; Musser, Melissa A; Walter, Teagan J; Labosky, Patricia A

2011-01-15

Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of insulin-expressing cells and insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic insulin granules and the presence of abnormal granules in insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation. Copyright © 2010 Elsevier Inc. All rights reserved.

Integrated transcriptomic and proteomic profiling of white spruce stems during the transition from active growth to dormancy.

PubMed

Galindo González, Leonardo M; El Kayal, Walid; Ju, Chelsea J-T; Allen, Carmen C G; King-Jones, Susanne; Cooke, Janice E K

2012-04-01

In the autumn, stems of woody perennials such as forest trees undergo a transition from active growth to dormancy. We used microarray transcriptomic profiling in combination with a proteomics analysis to elucidate processes that occur during this growth-to-dormancy transition in a conifer, white spruce (Picea glauca[Moench] Voss). Several differentially expressed genes were likely associated with the developmental transition that occurs during growth cessation in the cambial zone and the concomitant completion of cell maturation in vascular tissues. Genes encoding for cell wall and membrane biosynthetic enzymes showed transcript abundance patterns consistent with completion of cell maturation, and also of cell wall and membrane modifications potentially enabling cells to withstand the harsh conditions of winter. Several differentially expressed genes were identified that encoded putative regulators of cambial activity, cell development and of the photoperiodic pathway. Reconfiguration of carbon allocation figured centrally in the tree's overwintering preparations. For example, genes associated with carbon-based defences such as terpenoids were down-regulated, while many genes associated with protein-based defences and other stress mitigation mechanisms were up-regulated. Several of these correspond to proteins that were accumulated during the growth-to-dormancy transition, emphasizing the importance of stress protection in the tree's adaptive response to overwintering. © 2011 Blackwell Publishing Ltd.

Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

PubMed

Obata, Yuuki; Kimura, Shunsuke; Nakato, Gaku; Iizuka, Keito; Miyagawa, Yurika; Nakamura, Yutaka; Furusawa, Yukihiro; Sugiyama, Machiko; Suzuki, Keiichiro; Ebisawa, Masashi; Fujimura, Yumiko; Yoshida, Hisahiro; Iwanaga, Toshihiko; Hase, Koji; Ohno, Hiroshi

2014-12-01

Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE. © 2014 The Authors.

The differentiation directions of the bone marrow stromal cells under modeling microgravity

NASA Astrophysics Data System (ADS)

Nesterenko, Olga; Rodionova, Natalia; Katkova, Olena

Within experiments on rats simulating microgravity by base load remove from back limbs (duration of the experiment 1,5 months) on marrow stromal cells cultures (ex vivo, in vitro) comprising osteogenic cells-predecessors, extracted from femurs, studied their peculiarities of the colony formation ablity, the cell structure, some cytological and ultra-structural characteristics and differentiation direction. It was found that that under microgravity conditions there is a decline of the stromal cells colony formation intensity, decrease of the colonies size and cells mitotic activity that indicates decrease of their growth potential. Both in control and in experiment the colonies were presented by population of low-differentiated cells, differentiated cells and mature cells. The comparative cytological and morphometric analysis have shown that the studied stromal cells in colonies have the smaller sizes, more elongated shape, and higher nucleocytoplasmic ratio. Cells composition in the experiment colonies is reliably different by the ratio of the low-differentiating to being differentiated cells; a ratio of low-differentiated to already differentiated cells; ratio of differentiated cells to total number of all cells. In comparison with control group, amount of the cells passed trough a differentiation stage and mature cells in colonies is decreased by 3 to 4 times. Among the differentiated stromal cells in colonies increasing amount of adipocytes was revealed. The analysis of electron microscope microphotographs showed that in osteogenic cells differentiated under microgravity conditions, there is a reduction of the specific volume of a granular endoplasmic reticulum, Golgi's complex and quantity of nuclei reduction that indicates depression of the specific biosyntheses process intensity in cells. The increase of lysosomes and myelinic structures quantity is linked to organelles partial reduction. Consolidation of mitochondrias is an evidence of the cells’ energy metabolism disorder. In differentiated cells, disorganization and a cytoskeleton destruction was observed. Results showed that under microgravity conditions proliferative and differentiation (including osteogenic) potentialities of low-differentiated marrow stromal cells decreased, induction of their adipocytic differentiation was observes as well. Obtained results make a new contribution into gravitation sensitivity mechanisms understanding for stromal cells of the bone marrow which contain osteogenic cells- predecessors, features of the osteoporosis development.

The effects of restricted glycolysis on stem-cell like characteristics of breast cancer cells

PubMed Central

Banerjee, Arindam; Arvinrad, Pardis; Darley, Matthew; Laversin, Stéphanie A.; Parker, Rachel; Rose-Zerilli, Matthew J.J.; Townsend, Paul A.; Cutress, Ramsey I.; Beers, Stephen A.; Houghton, Franchesca D.; Birts, Charles N.; Blaydes, Jeremy P.

2018-01-01

Altered glycolysis is a characteristic of many cancers, and can also be associated with changes in stem cell-like cancer (SCLC) cell populations. We therefore set out to directly examine the effect of glycolysis on SCLC cell phenotype, using a model where glycolysis is stably reduced by adapting the cells to a sugar source other than glucose. Restricting glycolysis using this approach consistently resulted in cells with increased oncogenic potential; including an increase in SCLC cells, proliferation in 3D matrigel, invasiveness, chemoresistance, and altered global gene expression. Tumorigenicity in vivo was also markedly increased. SCLC cells exhibited increased dependence upon alternate metabolic pathways. They also became c-KIT dependent, indicating that their apparent state of maturation is regulated by glycolysis. Single-cell mRNA sequencing identified altered networks of metabolic-, stem- and signaling- gene expression within SCLC-enriched populations in response to glycolytic restriction. Therefore, reduced glycolysis, which may occur in niches within tumors where glucose availability is limiting, can promote tumor aggressiveness by increasing SCLC cell populations, but can also introduce novel, potentially exploitable, vulnerabilities in SCLC cells. PMID:29796188

Differential inhibition onto developing and mature granule cells generates high-frequency filters with variable gain

PubMed Central

Pardi, María Belén; Ogando, Mora Belén; Schinder, Alejandro F; Marin-Burgin, Antonia

2015-01-01

Adult hippocampal neurogenesis provides the dentate gyrus with heterogeneous populations of granule cells (GC) originated at different times. The contribution of these cells to information encoding is under current investigation. Here, we show that incoming spike trains activate different populations of GC determined by the stimulation frequency and GC age. Immature GC respond to a wider range of stimulus frequencies, whereas mature GC are less responsive at high frequencies. This difference is dictated by feedforward inhibition, which restricts mature GC activation. Yet, the stronger inhibition of mature GC results in a higher temporal fidelity compared to that of immature GC. Thus, hippocampal inputs activate two populations of neurons with variable frequency filters: immature cells, with wide‐range responses, that are reliable transmitters of the incoming frequency, and mature neurons, with narrow frequency response, that are precise at informing the beginning of the stimulus, but with a sparse activity. DOI: http://dx.doi.org/10.7554/eLife.08764.001 PMID:26163657

Critical role for invariant chain in CD1d-mediated selection and maturation of Vα14-invariant NKT cells.

PubMed

Sillé, Fenna C M; Martin, Constance; Jayaraman, Pushpa; Rothchild, Alissa; Besra, Gurdyal S; Behar, Samuel M; Boes, Marianne

2011-09-30

The development and maturation of Vα14 invariant (i)NKT cells in mice requires CD1d-mediated lipid antigen presentation in the thymus and the periphery. Cortical thymocytes mediate positive selection, while professional APCs are involved in thymic negative selection and in terminal maturation of iNKT cells in the periphery. CD1d requires entry in the endosomal pathway to allow antigen acquisition for assembly as lipid/CD1d complexes for display to iNKT cells. This process involves tyrosine-based sorting motifs in the CD1d cytoplasmic tail and invariant chain (Ii) that CD1d associates with in the endoplasmic reticulum. The function of Ii in iNKT cell thymic development and peripheral maturation had not been fully understood. Using mice deficient in Ii and the Ii-processing enzyme cathepsin S (catS), we addressed this question. Ii(-/-) mice but not catS(-/-) mice developed significantly fewer iNKT cells in thymus, that were less mature as measured by CD44 and NK1.1 expression. Ii(-/-) mice but not catS(-/-) mice developed fewer Vβ7(+) cells in their iNKT TCR repertoire than WT counterparts, indicative of a change in endogenous glycolipid antigen/CD1d-mediated iNKT cell selection. Finally, using a Mycobacterium tuberculosis infection model in macrophages, we show that iNKT developed in Ii(-/-) but not catS(-/-) mice have defective effector function. Our data support a role for professional APCs expressing Ii, but no role for catS in the thymic development and peripheral terminal maturation of iNKT cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

PubMed

Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

2015-05-01

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses.

PubMed

Wu, Gregory F; Corbo, Evann; Schmidt, Michelle; Smith-Garvin, Jennifer E; Riese, Matthew J; Jordan, Martha S; Laufer, Terri M; Brown, Eric J; Maltzman, Jonathan S

2011-07-01

The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T-cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP-76 protein in thymocytes. Here, we show that following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR-generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T-cell-mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T-cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nrf2 suppresses the function of dendritic cells to facilitate the immune escape of glioma cells.

PubMed

Wang, Jialiang; Liu, Peng; Xin, Shaoyan; Wang, Zongbao; Li, Jun

2017-11-15

Nrf2 is presented in dendritic cells (DCs) and contributes to the maintenance of redox homeostasis. However, the expression pattern and function of Nrf2 in the maturation of DCs in the glioma-infiltrated microenvironment remain unrevealed. Our study aims to investigate the roles of Nrf2 in glioma cell-tamed DCs and their impact on the downstream T cell proliferation and cytotoxicity to glioma cells. It was showed that the inducible maturation of DCs was significantly suppressed after stimulation with tumor-conditioned medium (TCM) prepared from glioma cells (LN-18 and U118MG), as suggested by the decreased CD80, CD86 and IL-12 p70 expression and higher levels of IL-10 than the normal astrocyte medium treated DCs. Moreover, the TCM-exposed DCs had significantly increased expression and transcriptional activity of Nrf2 compared to the negative control. Nrf2 inhibition in DC cells substantially antagonized the inhibitory effects of TCM on the maturation and activation of DC cells, reflected by the elevated maturation markers and IL-12 p70. We further confirmed that Nrf2 inhibition in TCM-exposed DC cells promoted the proliferation of T cells as evaluated by the CFSE-labeled assay and Th1 response shown by the elevated production of IFN-γ. The cytotoxic T lymphocyte assay revealed that Nrf2 genetic suppression in DC cells greatly enhanced the capacity of T cells in the cytotoxicity to glioma cells dependent on the E:T ratio. Collectively, our study demonstrated that Nrf2 inhibition in DCs in glioma-exposed microenvironment could enhance the maturation of DCs and the subsequent activation of T cells and their cytotoxicity on glioma cells. Copyright © 2017. Published by Elsevier Inc.

Modulation of Ocular Inflammation by Mesenchymal Stem Cells

DTIC Science & Technology

2017-03-01

mature myeloid cells in 64 host defense and resolution of inflammation, excessive innate immune response can have 65 deleterious effects on tissue...that MSCs can regulate 69 functions of mature innate immune cells , including polarization of inflammatory macrophages 70 into an anti-inflammatory... cells 191 As immune cells are primarily developed in lymphoid organs, single cell suspensions from bone 192 marrow, spleen, and submandibular lymph

ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs.

PubMed

Lee, Michelle H; Goralczyk, Anna G; Kriszt, Rókus; Ang, Xiu Min; Badowski, Cedric; Li, Ying; Summers, Scott A; Toh, Sue-Anne; Yassin, M Shabeer; Shabbir, Asim; Sheppard, Allan; Raghunath, Michael

2016-02-17

Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced 'browning' in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow.

Blocking the Maturation of OncomiRNAs Using pri-miRNA-17∼92 Aptamer in Retinoblastoma

PubMed Central

Subramanian, Nithya; Kanwar, Jagat R.; Kanwar, Rupinder K.

2015-01-01

The miR-17∼92. or oncomiR-1, cluster encodes oncogenic microRNAs (miRNAs), and it also promotes retinoblastoma (RB) tumor formation. Antagomir and miRNA mimics based approaches are widely tried against oncogenic and tumor suppressive miRNAs. Other methods for targeting cancer related miRNAs are still under development. In the current study, we focused on the pri-miRNA-17∼92 aptamer (pri-apt), which can potentially replace the mix of five antagomirs by one aptamer that function to abrogate the maturation of miR-17, miR-18a, and miR-19b (P<0.05) for targeting RB. We used RB cell lines WERI-Rb1 and Y79 as an in vitro model. Cellular changes upon transfecting the pri-apt led to S-phase arrest in WERI-Rb1 cells and onset of apoptosis in both Y79 and WERI-Rb1 cell lines. There was increased cytotoxicity as measured by lactate dehydrogenase activity in pri-apt treated Y79 cells (P<0.05), and significant inhibition of cell proliferation was observed in both of the cell lines. Thus we showed the antiproliferative property of pri-apt in RB cell lines, which can be readily modified by developing appropriate vectors for the delivery of the aptamer specifically to cancer cells. PMID:25513843

Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers.

PubMed

Li, Junjun; Minami, Itsunari; Yu, Leqian; Tsuji, Kiyotaka; Nakajima, Minako; Qiao, Jing; Suzuki, Masato; Shimono, Ken; Nakatsuji, Norio; Kotera, Hitetoshi; Liu, Li; Chen, Yong

2016-01-01

Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays.

Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers

PubMed Central

Minami, Itsunari; Yu, Leqian; Nakajima, Minako; Qiao, Jing; Shimono, Ken; Nakatsuji, Norio; Kotera, Hitetoshi; Chen, Yong

2016-01-01

Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays. PMID:27446217

RNAi as a Routine Route Toward Breast Cancer Therapy

DTIC Science & Technology

2014-05-01

hematopoietic stem/ progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with...Hematopoietic Cells (A and B) Genome browser tracks depict methylation profiles across a lymphoid (A) and myeloid (B) specific locus in blood cells ...multipotent populations, and two derived, mature cell types from the lymphoid and myeloid lineages, respectively. For comparison, we generated methylomes

Newly divided eosinophils limit ozone-induced airway hyperreactivity in nonsensitized guinea pigs

PubMed Central

Jacoby, David B.

2017-01-01

Ozone causes vagally mediated airway hyperreactivity and recruits inflammatory cells, including eosinophils, to lungs, where they mediate ozone-induced hyperreactivity 1 day after exposure but are paradoxically protective 3 days later. We aimed to test the role of newly divided eosinophils in ozone-induced airway hyperreactivity in sensitized and nonsensitized guinea pigs. Nonsensitized and sensitized guinea pigs were treated with 5-bromo-2-deoxyuridine (BrdU) to label newly divided cells and were exposed to air or ozone for 4 h. Later (1 or 3 days later), vagally induced bronchoconstriction was measured, and inflammatory cells were harvested from bone marrow, blood, and bronchoalveolar lavage. Ozone induced eosinophil hematopoiesis. One day after ozone, mature eosinophils dominate the inflammatory response and potentiate vagally induced bronchoconstriction. However, by 3 days, newly divided eosinophils have reached the lungs, where they inhibit ozone-induced airway hyperreactivity because depleting them with antibody to IL-5 or a TNF-α antagonist worsened vagally induced bronchoconstriction. In sensitized guinea pigs, both ozone-induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs 3 days later failed to occur. Thus mature eosinophils dominated the ozone-induced inflammatory response in sensitized guinea pigs. Depleting these mature eosinophils prevented ozone-induced airway hyperreactivity in sensitized animals. Ozone induces eosinophil hematopoiesis and recruitment to lungs, where 3 days later, newly divided eosinophils attenuate vagally mediated hyperreactivity. Ozone-induced hematopoiesis of beneficial eosinophils is blocked by a TNF-α antagonist or by prior sensitization. In these animals, mature eosinophils are associated with hyperreactivity. Thus interventions targeting eosinophils, although beneficial in atopic individuals, may delay resolution of airway hyperreactivity in nonatopic individuals. PMID:28258108

Newly divided eosinophils limit ozone-induced airway hyperreactivity in nonsensitized guinea pigs.

PubMed

Wicher, Sarah A; Jacoby, David B; Fryer, Allison D

2017-06-01

Ozone causes vagally mediated airway hyperreactivity and recruits inflammatory cells, including eosinophils, to lungs, where they mediate ozone-induced hyperreactivity 1 day after exposure but are paradoxically protective 3 days later. We aimed to test the role of newly divided eosinophils in ozone-induced airway hyperreactivity in sensitized and nonsensitized guinea pigs. Nonsensitized and sensitized guinea pigs were treated with 5-bromo-2-deoxyuridine (BrdU) to label newly divided cells and were exposed to air or ozone for 4 h. Later (1 or 3 days later), vagally induced bronchoconstriction was measured, and inflammatory cells were harvested from bone marrow, blood, and bronchoalveolar lavage. Ozone induced eosinophil hematopoiesis. One day after ozone, mature eosinophils dominate the inflammatory response and potentiate vagally induced bronchoconstriction. However, by 3 days, newly divided eosinophils have reached the lungs, where they inhibit ozone-induced airway hyperreactivity because depleting them with antibody to IL-5 or a TNF-α antagonist worsened vagally induced bronchoconstriction. In sensitized guinea pigs, both ozone-induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs 3 days later failed to occur. Thus mature eosinophils dominated the ozone-induced inflammatory response in sensitized guinea pigs. Depleting these mature eosinophils prevented ozone-induced airway hyperreactivity in sensitized animals. Ozone induces eosinophil hematopoiesis and recruitment to lungs, where 3 days later, newly divided eosinophils attenuate vagally mediated hyperreactivity. Ozone-induced hematopoiesis of beneficial eosinophils is blocked by a TNF-α antagonist or by prior sensitization. In these animals, mature eosinophils are associated with hyperreactivity. Thus interventions targeting eosinophils, although beneficial in atopic individuals, may delay resolution of airway hyperreactivity in nonatopic individuals. Copyright © 2017 the American Physiological Society.

Proanthocyanidins polymeric tannin from Stryphnodendron adstringens are active against Candida albicans biofilms.

PubMed

Luiz, Raul Leal Faria; Vila, Taissa Vieira Machado; de Mello, João Carlos Palazzo; Nakamura, Celso Vataru; Rozental, Sonia; Ishida, Kelly

2015-03-19

Biofilm formation is important in Candida albicans pathogenesis and constitutes a mechanism of antifungal resistance. Thus, we evaluated the effect of proanthocyanidins polymer-rich fractions from Stryphnodendron adstringens (fraction F2 and subfraction F2.4) against C. albicans biofilms. Firstly, the antifungal activity of F2 and F2.4 against planktonic cells of Candida albicans (ATCC 10231) was determined using broth microdilution method. Anti-biofilm effect of F2 and F2.4 was evaluated during biofilm formation or on mature biofilm of C. albicans and compared with standard antifungals amphotericin B and fluconazole. Metabolic activity of sessile and dispersion cells from biofilms after antifungal treatments were measured using a tetrazolium reduction assay and the biofilm total biomass was quantified by crystal violet-based assay. Morphological alterations after treatments were observed using scanning electron microscopy. The anti-biofilm effect of F2 and F2.4 were comparable to standard antifungals (amphotericin B and fluconazole). F2 and F2.4 treatments reduced biofilm metabolic activity (in sessile and in dispersion cells) during biofilm formation, and in mature biofilms, unlike fluconazole, which only prevents the biofilm formation. Treatments with F2, F2.4 or fluconazole reduced biofilm biomass during biofilm formation, but not in mature biofilm. Amphotericin B presented higher inhibitory effect on biofilm formation and on mature biofilm of C. albicans. F2 and F2.4 treatments led to the appearance of dumbbell-shaped blastoconidia and of blastoconidia clusters in biofilms. Proanthocyanidins polymer-rich fractions from S. adstringens successfully inhibited C. albicans planktonic growth and biofilm development, and they represent a potential new agent for the treatment of biofilm-associated candidiasis.

[Regulation of immune responses by exosomes derived from antigen presenting cells].

PubMed

Maravillas-Montero, José Luis; Martínez-Cortés, Ismael

2017-01-01

Cells release several biomolecules to the extracellular environment using them as a communication alternative with neighbor cells. Besides these molecules, cells also release more complex elements, like vesicles; structures composed of a lipidic bilayer with transmembrane proteins that protect a hydrophilic content. Exosomes are a small subtype of vesicles (30-150 nm), produced by many cell types, such as tumor cells, neurons, epithelial cells and immune cells. Included in this last group, antigen presenting cells produce exosomes that contain different types of molecules depending on their activation and/or maturation state. In recent years there has been an exponential interest in exosomes due to the recent evidences that show the immunomodulatory properties of these vesicles and therefore, their great potential in diagnostic approaches and development of therapies for different inflammation-associated pathologies.

Divergent regeneration‐competent cells adopt a common mechanism for callus initiation in angiosperms

PubMed Central

Hu, Bo; Zhang, Guifang; Liu, Wu; Shi, Jianmin; Wang, Hua; Qi, Meifang; Li, Jiqin; Qin, Peng; Ruan, Ying; Huang, Hai; Zhang, Yijing

2017-01-01

Abstract In tissue culture, the formation of callus from detached explants is a key step in plant regeneration; however, the regenerative abilities in different species are variable. While nearly all parts of organs of the dicot Arabidopsis thaliana are ready for callus formation, mature regions of organs in monocot rice (Oryza sativa) and other cereals are extremely unresponsive to tissue culture. Whether there is a common molecular mechanism beyond these different regenerative phenomena is unclear. Here we show that the Arabidopsis and rice use different regeneration‐competent cells to initiate callus, whereas the cells all adopt WUSCHEL‐RELATED HOMEOBOX 11 (WOX11) and WOX5 during cell fate transition. Different from Arabidopsis which maintains regeneration‐competent cells in mature organs, rice exhausts those cells during organ maturation, resulting in regenerative inability in mature organs. Our study not only explains this old perplexity in agricultural biotechnology, but also provides common molecular markers for tissue culture of different angiosperm species. PMID:28975033

Divergent regeneration-competent cells adopt a common mechanism for callus initiation in angiosperms.

PubMed

Hu, Bo; Zhang, Guifang; Liu, Wu; Shi, Jianmin; Wang, Hua; Qi, Meifang; Li, Jiqin; Qin, Peng; Ruan, Ying; Huang, Hai; Zhang, Yijing; Xu, Lin

2017-06-01

In tissue culture, the formation of callus from detached explants is a key step in plant regeneration; however, the regenerative abilities in different species are variable. While nearly all parts of organs of the dicot Arabidopsis thaliana are ready for callus formation, mature regions of organs in monocot rice ( Oryza sativa ) and other cereals are extremely unresponsive to tissue culture. Whether there is a common molecular mechanism beyond these different regenerative phenomena is unclear. Here we show that the Arabidopsis and rice use different regeneration-competent cells to initiate callus, whereas the cells all adopt WUSCHEL-RELATED HOMEOBOX 11 ( WOX11 ) and WOX5 during cell fate transition. Different from Arabidopsis which maintains regeneration-competent cells in mature organs, rice exhausts those cells during organ maturation, resulting in regenerative inability in mature organs. Our study not only explains this old perplexity in agricultural biotechnology, but also provides common molecular markers for tissue culture of different angiosperm species.

Proapoptotic BIM Impacts B Lymphoid Homeostasis by Limiting the Survival of Mature B Cells in a Cell-Autonomous Manner.

PubMed

Liu, Rui; King, Ashleigh; Bouillet, Philippe; Tarlinton, David M; Strasser, Andreas; Heierhorst, Jörg

2018-01-01

The proapoptotic BH3-only protein BIM ( Bcl2l11 ) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre ) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre ) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim -deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro , conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc -driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions.

Proapoptotic BIM Impacts B Lymphoid Homeostasis by Limiting the Survival of Mature B Cells in a Cell-Autonomous Manner

PubMed Central

Liu, Rui; King, Ashleigh; Bouillet, Philippe; Tarlinton, David M.; Strasser, Andreas; Heierhorst, Jörg

2018-01-01

The proapoptotic BH3-only protein BIM (Bcl2l11) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim-deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro, conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc-driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions. PMID:29623080

The effects of exercise and stress on the survival and maturation of adult-generated granule cells

PubMed Central

Snyder, Jason S.; Glover, Lucas R.; Sanzone, Kaitlin M.; Kamhi, J. Frances; Cameron, Heather A.

2009-01-01

Stress strongly inhibits proliferation of granule cell precursors in the dentate gyrus, while voluntary running has the opposite effect. Few studies, however, have examined the possible effects of these environmental manipulations on the maturation and survival of young granule cells. We examined number of surviving granule cells and the proportion of young neurons that were functionally mature, as defined by seizure-induced immediate-early gene expression, in 14 and 21 day-old granule cells in mice that were given access to a running wheel, restrained daily for 2 hours, or given no treatment during this period. Importantly, treatments began two days after BrdU injection, to isolate effects on survival from those on cell proliferation. We found a large increase in granule cell survival in running mice compared with controls at both time points. In addition, running increased the proportion of granule cells expressing the immediate-early gene Arc in response to seizures, suggesting that it speeds incorporation into circuits, i.e., functional maturation. Stressed mice showed no change in Arc expression, compared to control animals, but, surprisingly, showed a transient increase in survival of 14-day-old granule cells, which was gone 7 days later. Examination of cell proliferation, using the endogenous mitotic marker proliferating cell nuclear antigen (PCNA) showed an increase in cell proliferation after 12 days of running but not after 19 days of running. The number of proliferating cells was unchanged 24 hours after the 12th or 19th episode of daily restraint stress. These findings demonstrate that running has strong effects on survival and maturation of young granule cells as well as their birth and that stress can have positive but short-lived effects on granule cell survival. PMID:19156854

Ontogenetic characterization of sporangium and spore of Huperzia serrata: an anti-aging disease fern.

PubMed

Long, Hua; Li, Jing; Li, You-You; Xie, De-Yu; Peng, Qing-Zhong; Li, Li

2016-12-01

Huperzia serrata is a medicinal plant used in Traditional Chinese Medicine, which has been used to prevent against aging diseases. It is mainly propagated by spores and grows extremely slowly. Due to severe harvest, it is a highly endangered species. In this report, we characterize ontogenesis of sporangia and spores that are associated with propagation. A wild population of H. serrata plants is localized in western Hunan province, China and protected by Chinese Government to study its development (e.g. sporangia and spores) and ecology. Both field and microscopic observations were conducted for a few of years. The development of sporangia from their initiation to maturation took nearly 1 year. Microscopic observations showed that the sporangial walls were developed from epidermal cells via initiation, cell division, and maturation. The structure of the mature sporangial wall is composed of one layer of epidermis, two middle layers of cells, and one layer of tapetum. Therefore, the sporangium is the eusporangium type. Spore development is characterized into six stages, initiation from epidermal cell and formation of sporogenous cells, primary sporogenous cell, secondary sporogenous cell, spore mother cell, tetrad, and maturation. The sporangial development of H. serrata belongs to the eusporangium type. The development takes approximately 1 year period from the initiation to the maturation. These data are useful for improving propagation of this medicinal plant in the future.

The potential role of ribosomal protein S5 on cell cycle arrest and initiation of murine erythroleukemia cell differentiation.

PubMed

Matragkou, Christina N; Papachristou, Eleni T; Tezias, Sotirios S; Tsiftsoglou, Asterios S; Choli-Papadopoulou, Theodora; Vizirianakis, Ioannis S

2008-07-01

Evidence now exists to indicate that some ribosomal proteins besides being structural components of the ribosomal subunits are involved in the regulation of cell differentiation and apoptosis. As we have shown earlier, initiation of erythroid differentiation of murine erythroleukemia (MEL) cells is associated with transcriptional inactivation of genes encoding ribosomal RNAs and ribosomal proteins S5 (RPS5) and L35a. In this study, we extended these observations and investigated whether transfection of MEL cells with RPS5 cDNA affects the onset of initiation of erythroid maturation and their entrance in cell cycle arrest. Stably transfected MEL cloned cells (MEL-C14 and MEL-C56) were established and assessed for their capacity to produce RPS5 RNA transcript and its translated product. The impact of RPS5 cDNA transfection on the RPS5 gene expression patterns and the accumulation of RPS5 protein in inducible transfected MEL cells were correlated with their ability to: (a) initiate differentiation, (b) enter cell cycle arrest at G(1)/G(0) phase, and (c) modulate the level of cyclin-dependent kinases CDK2, CDK4, and CDK6. The data presented indicate that deregulation of RPS5 gene expression (constitutive expression) affects RPS5 protein level and delays both the onset of initiation of erythroid maturation and entrance in cell cycle arrest in inducer-treated MEL cells. 2008 Wiley-Liss, Inc.

Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kazama, Tomohiko; Fujie, Masaki; Endo, Tuyoshi

2008-12-19

We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin,more » followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.« less

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

PubMed Central

Rodríguez, Lleretny; Navarrete, Felipe I.; Tovar, Heribelt; Cox, José F.

2008-01-01

Purpose In order to simplify cloning, a new method that does not require micromanipulators was used. We aimed to evaluate the developmental potential of two bovine cell lines upon cloning. Materials and methods In vitro matured bovine oocytes, were released from zona pellucida, enucleated, fused to foetal or adult somatic donor cells. The reconstructed embryos were reprogrammed, activated and cultured until blastocyst stage. No micromanipulators were used. Blastocyst rate and quality was scored. Some expanded (d7) blastocysts were transferred to recipient cattle and collected back at d17 to assess elongation. Results High developmental potential in vitro of cloned embryos to expanded (d7) blastocysts was achieved (52.6%). In one cell line, 65.7% of blastocysts was scored. Most blastocysts (87.4%) were graded as excellent. In vivo development to elongation (day-17) in temporary recipient cows also showed a high developmental potential (11/18 transferred blastocysts elongated). Conclusions Hand-made cloning is an efficient alternative for cloning in cattle. PMID:18205035

Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation.

PubMed

Tanihara, Fuminori; Hirata, Maki; Nhien, Nguyen Thi; Hirano, Takayuki; Kunihara, Toshiki; Otoi, Takeshige

2018-05-16

The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100, and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system.

Long-Term Outcome of Administration of c-kitPOS Cardiac Progenitor Cells After Acute Myocardial Infarction: Transplanted Cells Do Not Become Cardiomyocytes, Structural and Functional Improvement and Proliferation of Endogenous Cells Persist for at Least One Year

PubMed Central

Tang, Xian-Liang; Li, Qianhong; Rokosh, Gregg; Sanganalmath, Santosh K.; Chen, Ning; Ou, Qinghui; Stowers, Heather; Hunt, Greg; Bolli, Roberto

2016-01-01

RATIONALE Cardiac progenitor cells (CPCs) improve left ventricular (LV) remodeling and function after acute or chronic myocardial infarction (MI). However, the long-term (>5 weeks) effects, potential tumorigenicity, and fate of transplanted CPCs are unknown. OBJECTIVE To assess the outcome of CPC therapy at 1 year. METHODS AND RESULTS Female rats underwent a 90-min coronary occlusion; 4 h after reperfusion, they received intracoronarily vehicle or 1 million male, syngeneic CPCs. One year later, CPC-treated rats exhibited smaller scars and more viable myocardium in the risk region, along with improved LV remodeling and regional and global LV function. No tumors were observed. Some transplanted (Y-chromosomePOS) CPCs (or their progeny) persisted and continued to proliferate, but they failed to acquire a mature cardiomyocyte phenotype and were too few (4-8% of nuclei) to account for the benefits of CPC therapy. Surprisingly, CPC transplantation triggered a prolonged proliferative response of endogenous cells, resulting in increased formation of endothelial cells and Y-chromosomeNEG CPCs for 12 months and increased formation, for at least 7 months, of small cells that expressed cardiomyocytic proteins (α-sarcomeric actin) but did not have a mature cardiomyocyte phenotype. CONCLUSIONS The beneficial effects of CPCs on LV remodeling and dysfunction are sustained for at least 1 year, and thus are likely to be permanent. Since transplanted CPCs do not differentiate into mature myocytes, their major mechanism of action must involve paracrine actions. These paracrine mechanisms could be very prolonged because some CPCs engraft, proliferate, and persist at 1 year. This is the first report that transplantation of any cell type in the heart induces a proliferative response that lasts at least 1 year. The results strongly support the safety and clinical utility of CPC therapy. PMID:26838790

The cancer paradigms of mammalian regeneration: can mammals regenerate as amphibians?

PubMed

Sarig, Rachel; Tzahor, Eldad

2017-04-01

Regeneration in mammals is restricted to distinct tissues and occurs mainly by expansion and maturation of resident stem cells. During regeneration, even subtle mutations in the proliferating cells may cause a detrimental effect by eliciting abnormal differentiation or malignant transformation. Indeed, cancer in mammals has been shown to arise through deregulation of stem cells maturation, which often leads to a differentiation block and cell transformation. In contrast, lower organisms such as amphibians retain a remarkable regenerative capacity in various organs, which occurs via de- and re-differentiation of mature cells. Interestingly, regenerating amphibian cells are highly resistant to oncogenic transformation. Therapeutic approaches to improve mammalian regeneration mainly include stem-cell transplantations; but, these have proved unsuccessful in non-regenerating organs such as the heart. A recently developed approach is to induce de-differentiation of mature cardiomyocytes using factors that trigger their re-entry into the cell cycle. This novel approach raises numerous questions regarding the balance between transformation and regeneration induced by de-differentiation of mature mammalian somatic cells. Can this balance be controlled artificially? Do de-differentiated cells acquire the protection mechanisms seen in regenerating cells of lower organisms? Is this model unique to the cardiac tissue, which rarely develops tumors? This review describes regeneration processes in both mammals and lower organisms and, particularly, the ability of regenerating cells to avoid transformation. By comparing the characteristics of mammalian embryonic and somatic cells, we discuss therapeutic strategies of using various cell populations for regeneration. Finally, we describe a novel cardiac regeneration approach and its implications for regenerative medicine. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

PubMed

Cheng, Catherine; Nowak, Roberta B; Biswas, Sondip K; Lo, Woo-Kuen; FitzGerald, Paul G; Fowler, Velia M

2016-08-01

To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein. We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers. F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers. These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.

CD40L+ CD4+ memory T cells migrate in a CD62P-dependent fashion into reactive lymph nodes and license dendritic cells for T cell priming

PubMed Central

Martín-Fontecha, Alfonso; Baumjohann, Dirk; Guarda, Greta; Reboldi, Andrea; Hons, Miroslav; Lanzavecchia, Antonio; Sallusto, Federica

2008-01-01

There is growing evidence that the maturation state of dendritic cells (DCs) is a critical parameter determining the balance between tolerance and immunity. We report that mouse CD4+ effector memory T (TEM) cells, but not naive or central memory T cells, constitutively expressed CD40L at levels sufficient to induce DC maturation in vitro and in vivo in the absence of antigenic stimulation. CD4+ TEM cells were excluded from resting lymph nodes but migrated in a CD62P-dependent fashion into reactive lymph nodes that were induced to express CD62P, in a transient or sustained fashion, on high endothelial venules. Trafficking of CD4+ TEM cells into chronic reactive lymph nodes maintained resident DCs in a mature state and promoted naive T cell responses and experimental autoimmune encephalomyelitis (EAE) to antigens administered in the absence of adjuvants. Antibodies to CD62P, which blocked CD4+ TEM cell migration into reactive lymph nodes, inhibited DC maturation, T cell priming, and induction of EAE. These results show that TEM cells can behave as endogenous adjuvants and suggest a mechanistic link between lymphocyte traffic in lymph nodes and induction of autoimmunity. PMID:18838544

Proteomic Analyses of the Effects of Drugs of Abuse on Monocyte-Derived Mature Dendritic Cells

PubMed Central

Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikunar; Nair, B.; Sykes, Donald E.; Schwartz, Stanley A.

2010-01-01

Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that alter normal biological processes when monocyte-derived mature dendritic cells were treated with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins including those that modulate apoptosis, protein folding, protein kinase activity, and metabolism and proteins that function as intracellular signal transduction molecules. Proteomic data were validated using a combination of quantitative, real-time PCR and Western blot analyses. These studies will help to identify the molecular mechanisms, including the expression of several functionally important classes of proteins that have emerged as potential mediators of pathogenesis. These proteins may predispose immunocompetent cells, including dendritic cells, to infection with viruses such as HCV and HIV-1, which are associated with drug abuse. PMID:19811410

Differences in cumulus cells gene expression between modified natural and stimulated in vitro fertilization cycles.

PubMed

Papler, Tanja Burnik; Bokal, Eda Vrtačnik; Tacer, Klementina Fon; Juvan, Peter; Virant Klun, Irma; Devjak, Rok

2014-01-01

The aim of our study was to determine whether there are any differences in the cumulus cell gene expression profile of mature oocytes derived from modified natural IVF and controlled ovarian hyperstimulation cycles and if these changes could help us understand why modified natural IVF has lower success rates. Cumulus cells surrounding mature oocytes that developed to morulae or blastocysts on day 5 after oocyte retrieval were submitted to microarray analysis. The obtained data were then validated using quantitative real-time PCR. There were 66 differentially expressed genes between cumulus cells of modified natural IVF and controlled ovarian hyperstimulation cycles. Gene ontology analysis revealed the oxidation-reduction process, glutathione metabolic process, xenobiotic metabolic process and gene expression were significantly enriched biological processes in MNIVF cycles. Among differentially expressed genes we observed a large group of small nucleolar RNA's whose role in folliculogenesis has not yet been established. The increased expression of genes involved in the oxidation-reduction process probably points to hypoxic conditions in modified natural IVF cycles. This finding opens up new perspectives for the establishment of the potential role that oxidation-reduction processes have in determining success rates of modified natural IVF.

Activation of chicken bone marrow-derived dendritic cells induced by a Salmonella Enteritidis ghost vaccine candidate.

PubMed

Kamble, N M; Jawale, C V; Lee, J H

2016-10-01

Bacterial Ghost-based vaccine development has been applied to a variety of gram-negative bacteria. Developed Salmonella Enteritidis (S. Enteritidis) ghost are promising vaccine candidates because of their immunogenic and enhanced biosafety potential. In this study, we aimed to evaluate the immunostimulatory effect of a S. Enteritidis ghost vaccine on the maturation of chicken bone marrow-derived dendritic cells (chBM-DCs) in vitro The immature chBM-DCs were stimulated with S. Enteritidis ghost vaccine candidate. The vaccine efficiently stimulated maturation events in chBM-DCs, indicated by up-regulated expression of CD40, CD80, and MHC-II molecules. Immature BM-DCs responded to stimulation with S. Enteritidis ghost by increased expression of IL-6 and IL-12p40 cytokines. Also, S. Enteritidis ghost stimulated chBM-DCs induced the significant expression of IFN-γ and IL-2 in co-cultured autologous CD4+ T cells. In conclusion, our data suggest that S. Enteritidis ghost vaccine candidate is capable of activating and interacting with chBM-DCs. The results from current study may help for rational designing of Salmonella ghost based heterologous antigen delivery platforms to dendritic cells. © 2016 Poultry Science Association Inc.

Postnatal development of the myenteric plexus in cat stomach.

PubMed

Lolova, I; Itsev, D

1983-01-01

The postnatal development of the myenteric plexus in cat stomach has been studied at birth, on the 14th, 30th, 45th and 180th postnatal days, using light- and electronmicroscopic methods. In newborn kittens the main network of the Auerbach plexus is well formed, but the myenteric ganglia are composed of nerve cells with different maturity and a scarce neuropile. During the first two postnatal weeks the dimensions of the ganglia increase owing to the increase of the nerve bodies and the rising number of glials cells and intercellular fibres. This is accompanied by a potentiation of the AChE-activity, mainly in the nerve cell bodies and to a lesser extent in the neuropile. Impregnation reveals different in calibre and form nerve fibres and terminals. Different ultrastructural types of neurones are identified on the 14th day. Later development is expressed in the formation of large compact ganglia and thick connecting strands. The number of AChE-positive fibres in the neuropile increases. Owing to the increase in the cell organelles and their more advanced maturity, it is possible to define the ultrastructural type of an ever increasing number of neurones.

Inhibition of HBV Replication in HepG2.2.15 Cells by Human Peripheral Blood Mononuclear Cell-Derived Dendritic Cells.

PubMed

Liu, Tao; Song, Hong-Li; Zheng, Wei-Ping; Shen, Zhong-Yang

2015-01-01

Anti-HBV therapy is essential for patients awaiting liver transplantation. This study aimed to explore the effects of dendritic cells (DCs) derived from the peripheral blood of hepatitis B patients on the replication of HBV in vivo and to evaluate the biosafety of DCs in clinical therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from HBV-infected patients and maturation-promoting factors and both HBsAg and HBcAg were used to induce DC maturation. Mature DCs and lymphocytes were co-cultured with human hepatocyte cell HL-7702 or HBV-producing human hepatocellular carcinoma cell HepG2.2.15. We found that mature lymphocytes exposed to DCs in vitro did not influence morphology or activities of HL-7702 and HepG2.2.15 cells. Liver function indexes and endotoxin levels in the cell supernatants did not change in these co-cultures. Additionally, supernatant and intracellular HBV DNA levels were reduced when HepG2.2.15 cells were co-cultured with mature lymphocytes that had been cultured with DCs, and HBV covalently closed circular DNA (cccDNA) levels in HepG2.2.15 cells also decreased. Importantly, DC-mediated immunotherapy had no mutagenic effect on HBV genomic DNA by gene sequencing of the P, S, X, and C regions of HBV genomic DNA. We conclude that PBMC-derived DCs from HBV-infected patients act on autologous lymphocytes to suppress HBV replication and these DC clusters showed favorable biosafety. © 2015 by the Association of Clinical Scientists, Inc.

Iκb Kinase α Is Essential for Mature B Cell Development and Function

PubMed Central

Kaisho, Tsuneyasu; Takeda, Kiyoshi; Tsujimura, Tohru; Kawai, Taro; Nomura, Fumiko; Terada, Nobuyuki; Akira, Shizuo

2001-01-01

IκB kinase (IKK) α and β phosphorylate IκB proteins and activate the transcription factor, nuclear factor (NF)-κB. Although both are highly homologous kinases, gene targeting experiments revealed their differential roles in vivo. IKKα is involved in skin and limb morphogenesis, whereas IKKβ is essential for cytokine signaling. To elucidate in vivo roles of IKKα in hematopoietic cells, we have generated bone marrow chimeras by transferring control and IKKα-deficient fetal liver cells. The mature B cell population was decreased in IKKα−/− chimeras. IKKα−/− chimeras also exhibited a decrease of serum immunoglobulin basal level and impaired antigen-specific immune responses. Histologically, they also manifested marked disruption of germinal center formation and splenic microarchitectures that depend on mature B cells. IKKα−/− B cells not only showed impairment of survival and mitogenic responses in vitro, accompanied by decreased, although inducible, NF-κB activity, but also increased turnover rate in vivo. In addition, transgene expression of bcl-2 could only partially rescue impaired B cell development in IKKα−/− chimeras. Taken together, these results demonstrate that IKKα is critically involved in the prevention of cell death and functional development of mature B cells. PMID:11181694

Stem cell metabolism in tissue development and aging

PubMed Central

Shyh-Chang, Ng; Daley, George Q.; Cantley, Lewis C.

2013-01-01

Recent advances in metabolomics and computational analysis have deepened our appreciation for the role of specific metabolic pathways in dictating cell fate. Once thought to be a mere consequence of the state of a cell, metabolism is now known to play a pivotal role in dictating whether a cell proliferates, differentiates or remains quiescent. Here, we review recent studies of metabolism in stem cells that have revealed a shift in the balance between glycolysis, mitochondrial oxidative phosphorylation and oxidative stress during the maturation of adult stem cells, and during the reprogramming of somatic cells to pluripotency. These insights promise to inform strategies for the directed differentiation of stem cells and to offer the potential for novel metabolic or pharmacological therapies to enhance regeneration and the treatment of degenerative disease. PMID:23715547

Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko

We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventionalmore » DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.« less

Maturation of sperm volume regulation in the rat epididymis

PubMed Central

Damm, Oliver S.; Cooper, Trevor G.

2010-01-01

Sperm maturation in the epididymis may involve differences between mature and immature spermatozoa in their volume regulatory osmolyte response. Spermatozoa obtained from the rat caput and cauda epididymidis were examined for their ability to regulate volume after transfer from in situ epididymal osmolality (measured to be 343 ± 13 and 365 ± 19 mmol kg−1, respectively) to that of the female tract in single- and multiple-step protocols. Cells withstood the single-step treatment better than the multistep protocol. Sperm volume estimates by flow cytometric measurements of forward scatter of cells with intact head membranes was more sensitive than those by assessing cell coiling microscopically. At osmolalites below 210 mmol kg−1 both caput and cauda cells ruptured, limiting the use of flow cytometry. Above this critical value, the use of quinine showed that both caput and cauda cells could regulate volume, but cauda cells were the more effective. Of several organic osmolytes studied, myo-inositol, glutamate and KCl caused only temporary and slight swelling of spermatozoa cells in hypotonic medium. Spermatozoa of both maturities seemed to use potassium as the preferred osmolyte for regulating volume. PMID:20531277

The interrelationship between bone and fat: from cellular see-saw to endocrine reciprocity.

PubMed

Sadie-Van Gijsen, H; Crowther, N J; Hough, F S; Ferris, W F

2013-07-01

The number of mature osteoblasts and marrow adipocytes in bone is influenced by the differentiation of the common mesenchymal progenitor cell towards one phenotype and away from the other. Consequently, factors which promote adipogenesis not only lead to fatty marrow but also inhibit osteoblastogenesis, resulting in decreased osteoblast numbers, diminished bone formation and, potentially, inadequate bone mass and osteoporosis. In addition to osteoblast and bone adipocyte numbers being influenced by this skewing of progenitor cell differentiation towards one phenotype, mature osteoblasts and adipocytes secrete factors which may evoke changes in the cell fate and function of each other. This review examines the endogenous factors, such as PPAR-γ2, Wnt, IGF-1, GH, FGF-2, oestrogen, the GP130 signalling cytokines, vitamin D and glucocorticoids, which regulate the selection between osteoblastogenesis and adipogenesis and the interrelationship between fat and bone. The role of adipokines on bone, such as adiponectin and leptin, as well as adipose-derived oestrogen, is reviewed and the role of bone as an energy regulating endocrine organ is discussed.

Reparative neurogenesis after cerebral ischemia: Clinical application prospects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khodanovich, M. Yu., E-mail: khodanovich@mail.tsu.ru

At the present time two main approaches are in the focus of neurobiological studies of brain recovery after a stroke. One of them is concerned with the infusion of stem cells in damaged brain. The second approach is directed at the stimulation of endogenous reparative processes, in particular, adult neurogenesis. This review considers alterations of adult neurogenesis caused by cerebral ischemia and possible pathways of its regulation. Multiple studies on animal models have shown that adult neurogenesis is mostly increased by cerebral ischemia. In spite of increasing proliferation and moving neural progenitors to infarct zone, most newborn neurons die beforemore » reaching maturity. Besides, an increase of neurogenesis in pathological conditions is mainly due to recruitment of new stem cells, but not due to an additional precursor-cells division that results in an overall decline of the regeneration capacity. Thus, the endogenous reparative mechanisms are not sufficient, and the search for new targets to promote proliferation, survival, and maturation of new neurons after a stroke is needed. Neurotransmitter systems and anti-inflammatory drugs are considered as potential regulators of post-ischemic neurogenesis growth factors.« less

Reparative neurogenesis after cerebral ischemia: Clinical application prospects

NASA Astrophysics Data System (ADS)

Khodanovich, M. Yu.

2015-11-01

At the present time two main approaches are in the focus of neurobiological studies of brain recovery after a stroke. One of them is concerned with the infusion of stem cells in damaged brain. The second approach is directed at the stimulation of endogenous reparative processes, in particular, adult neurogenesis. This review considers alterations of adult neurogenesis caused by cerebral ischemia and possible pathways of its regulation. Multiple studies on animal models have shown that adult neurogenesis is mostly increased by cerebral ischemia. In spite of increasing proliferation and moving neural progenitors to infarct zone, most newborn neurons die before reaching maturity. Besides, an increase of neurogenesis in pathological conditions is mainly due to recruitment of new stem cells, but not due to an additional precursor-cells division that results in an overall decline of the regeneration capacity. Thus, the endogenous reparative mechanisms are not sufficient, and the search for new targets to promote proliferation, survival, and maturation of new neurons after a stroke is needed. Neurotransmitter systems and anti-inflammatory drugs are considered as potential regulators of post-ischemic neurogenesis growth factors.

Mind bomb-1 is an essential modulator of long-term memory and synaptic plasticity via the Notch signaling pathway

PubMed Central

2012-01-01

Background Notch signaling is well recognized as a key regulator of the neuronal fate during embryonic development, but its function in the adult brain is still largely unknown. Mind bomb-1 (Mib1) is an essential positive regulator in the Notch pathway, acting non-autonomously in the signal-sending cells. Therefore, genetic ablation of Mib1 in mature neuron would give valuable insight to understand the cell-to-cell interaction between neurons via Notch signaling for their proper function. Results Here we show that the inactivation of Mib1 in mature neurons in forebrain results in impaired hippocampal dependent spatial memory and contextual fear memory. Consistently, hippocampal slices from Mib1-deficient mice show impaired late-phase, but not early-phase, long-term potentiation and long-term depression without change in basal synaptic transmission at SC-CA1 synapses. Conclusions These data suggest that Mib1-mediated Notch signaling is essential for long-lasting synaptic plasticity and memory formation in the rodent hippocampus. PMID:23111145

CD40 ligation and phagocytosis differently affect the differentiation of monocytes into dendritic cells.

PubMed

Rosenzwajg, Michelle; Jourquin, Frédéric; Tailleux, Ludovic; Gluckman, Jean Claude

2002-12-01

That monocytes can differentiate into macrophages or dendritic cells (DCs) makes them an essential link between innate and adaptive immunity. However, little is known about how interactions with pathogens or T cells influence monocyte engagement toward DCs. We approached this point in cultures where granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 induced monocytes to differentiate into immature DCs. Activating monocytes with soluble CD40 ligand (CD40L) led to accelerated differentiation toward mature CD83(+) DCs with up-regulated human leukocyte antigen-DR, costimulatory molecules and CD116 (GM-CSF receptor), and down-regulation of molecules involved in antigen capture. Monocytes primed by phagocytosis of antibody-opsonized, killed Escherichia coli differentiated into DCs with an immature phenotype, whereas Zymosan priming yielded active DCs with an intermediate phenotype. Accordingly, DCs obtained from cultures with CD40L or after Zymosan priming had a decreased capacity to endocytose dextran, but only DCs cultured with CD40L had increased capacity to stimulate allogeneic T cells. DCs obtained after E. coli or Zymosan priming of monocytes produced high levels of proinflammatory tumor necrosis factor alpha and IL-6 as well as of regulatory IL-10, but they produced IL-12p70 only after secondary CD40 ligation. Thus, CD40 ligation on monocytes accelerates the maturation of DCs in the presence of GM-CSF/IL-4, whereas phagocytosis of different microorganisms does not alter and even facilitates their potential to differentiate into immature or active DCs, the maturation of which can be completed upon CD40 ligation. In vivo, such differences may correspond to DCs with different trafficking and T helper cell-stimulating capacities that could differently affect induction of adaptive immune responses to infections.

Betalactam antibiotics affect human dendritic cells maturation through MAPK/NF-kB systems. Role in allergic reactions to drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Soledad; Department of Medical Biochemistry, Molecular Biology and Immunology, The University of Seville Medical School, Seville; Gomez, Enrique

The mechanisms leading to drug allergy in predisposed patients, especially those related to T-cell-mediated drug hypersensitivity, are not well understood. A key event in allergic reactions to drugs is the maturation process undergone by dendritic cells (DCs). Although amoxicillin (AX) has been reported to interact and maturate DCs from patients with AX-induced delayed-type hypersensitivity, the cell signaling pathways related to AX-mediated DC maturation have not been elucidated. We sought to determine the role of the MAPK and NF-κΒ pathways on AX-induced DC maturation and functional status. For that purpose, in monocyte-derived-DCs from AX-delayed allergic patients and tolerant subjects, we analyzedmore » the activation pattern of p38MAPK, JNK, and ERK signaling and the NF-κB, maturation markers as well as endocytosis and allostimulatory capacities driven by AX-stimulated-DCs. Our data reveal that AX induces an increase in the phosphorylation levels of the three MAPKsand activated NF-κB in DCs from allergic patients. Moreover, the inhibition of these pathways prevents the up-regulation of surface molecules induced by AX. Additionally, we observed that the allostimulatory capacity and the endocytosis down-regulation in AX-stimulated-DCs from allergic patients depend on JNK and NF-κB activities. Taken together, our data shed light for the first time on the main signaling pathways involved in DC maturation from AX-delayed allergic patient. - Highlights: • The cell signaling pathways related to drug-mediated DC maturation were tested. • Amoxicillin induces activation of MAPK and NF-κB in DCs from allergic patients. • The inhibition of these pathways prevents the up-regulation of DC surface molecules. • Their allostimulatory and endocytosis capacities depend on JNK and NF-κB activities. • The low involvement of p38-MAPK could be the cause of an incomplete DC maturation.« less